Alpha-amylase variants and polynucleotides encoding same

ABSTRACT

The present invention relates to alpha-amylase variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/002,576 filed Jun. 7, 2018, now U.S. Pat. No. 10,689,635, which is acontinuation of U.S. application Ser. No. 15/093,098 filed Apr. 7, 2016,now U.S. Pat. No. 10,017,752, which is a continuation of U.S.application Ser. No. 14/346,094 filed Mar. 20, 2014, now U.S. Pat. No.9,334,485, which is a 35 U.S.C. 371 national application ofPCT/EP2012/070575 filed Oct. 17, 2012, which claims priority or thebenefit under 35 U.S.C. 119 of European application no. 11185478.2 filedOct. 17, 2011 and U.S. provisional application No. 61/548,434 filed Oct.18, 2011. The content of these applications is fully incorporated hereinby reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to alpha-amylase variants, polynucleotidesencoding the variants, methods of producing the variants, and methods ofusing the variants.

Description of the Related Art

Alpha-amylases (E.C. 3.2.1.1) constitute a group of enzymes whichcatalyze hydrolysis of starch, glycogen and related polysaccharides andoligosaccharides.

Alpha-amylases are used commercially for a variety of purposes such asin the initial stages of starch processing (e.g., liquefaction); in wetmilling processes; and in alcohol production from carbohydrate sources.They are also used as cleaning agents or adjuncts in detergent matrices;in the textile industry for starch desizing; in baking applications; inthe beverage industry; in oil fields in drilling processes; in recyclingprocesses, e.g., for de-inking paper; and in animal feed.

Some commercial alpha-amylases for, e.g., starch liquefaction originatefrom Bacillus licheniformis or Bacillus stearothermophilus. Proteinengineered variants of wild type enzymes have been developed to overcomeprocess issues. There is still a need, though, for novel alpha-amylaseswith improved properties, such as higher stability at low pH, lowcalcium and high temperature. Such enzymes will allow the starchliquefaction process to be run at reduced pH which has a positiveinfluence on chemical savings.

It is an object of the present invention to provide novel alpha-amylasevariants having an increased stability at low pH and/or at hightemperature, in particular at low calcium concentrations.

SUMMARY OF THE INVENTION

The present inventors have found that alpha-amylase variants comprisinga substitution with proline at a position corresponding to position 185combined with a substitution at one or more positions corresponding topositions 15, 48, 49, 50, 107, 116, 133, 138, 156, 176, 181, 187, 188,190, 197, 201, 205, 209, 213, 239, 241, 255, 264, 299, 360, 375, 416,437, 474 and 475 of SEQ ID NO: 1 have an increased stability at low pHand/or at high temperature, in particular at low calcium concentrations.

The present invention therefore relates to alpha-amylase variantscomprising a substitution with proline at a position corresponding toposition 185 of SEQ ID NO: 1 and further comprising a substitution atone or more positions corresponding to positions 15, 48, 49, 50, 107,116, 133, 138, 156, 176, 181, 187, 188, 190, 197, 201, 205, 209, 213,239, 241, 255, 264, 299, 360, 375, 416, 437, 474 and 475 of SEQ ID NO:1, wherein the variant has at least 60% and less than 100% sequenceidentity to (i) the mature polypeptide of any of SEQ ID NOs: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or (ii) amino acids 1 to 483 of SEQID NO: 1, amino acids 1 to 483 of SEQ ID NO: 2, amino acids 1 to 485 ofSEQ ID NO: 3, amino acids 1 to 482 of SEQ ID NO: 4, amino acids 1 to 484of SEQ ID NO: 5, amino acids 1 to 483 of SEQ ID NO: 6, amino acids 1 to485 of SEQ ID NO: 7, amino acids 1 to 485 of SEQ ID NO: 8, amino acids 1to 485 of SEQ ID NO: 9, amino acids 1 to 485 of SEQ ID NO: 10, aminoacids 1 to 485 of SEQ ID NO: 11, amino acids 1 to 480 of SEQ ID NO: 12,amino acids 1 to 483 of SEQ ID NO: 13 or amino acids 1 to 481 of SEQ IDNO: 14, and wherein the variant has alpha-amylase activity.

The present invention also relates to isolated polynucleotides encodingthe variants; nucleic acid constructs, vectors, and host cellscomprising the polynucleotides; and methods of producing the variants.

The present invention also relates to uses of the variants of theinvention and to a method of producing liquefied starch and a method ofproducing a fermentation product.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C and 1D show an alignment of alpha-amylases with theamino acid sequences of:

SEQ ID NO: 1 is a Bacillus licheniformis alpha-amylase.

SEQ ID NO: 2 is a Bacillus stearothermophilus alpha-amylase.

SEQ ID NO: 3 is the Bacillus alpha-amylase TS-23 described in J. Appl.Microbiology, 1997, 82: 325-334 (SWALL:q59222).

SEQ ID NO: 4 is Bacillus flavothermus alpha-amylase AMY1048 described inWO 2005/001064.

SEQ ID NO: 5 is Bacillus alpha-amylase TS-22 described as SEQ ID NO: 21in WO 04/113511.

SEQ ID NO: 6 is a Bacillus amyloliquefaciens alpha-amylase.

SEQ ID NO: 7 is Bacillus alkaline sp. SP690 amylase described as SEQ IDNO 1 in WO 95/26397.

SEQ ID NO: 8 is Bacillus halmapalus alpha-amylase described as SEQ ID NO2 in WO 95/26397.

SEQ ID NO: 9 is Bacillus alkaline sp. AA560 amylase described as SEQ IDNO 4 in WO 00/60060.

SEQ ID NO: 10 is Bacillus alkaline sp. A 7-7 amylase described as SEQ IDNO 2 in WO200210356.

SEQ ID NO: 11 is Bacillus alkaline sp. SP707 amylase described inTsukamoto et al., 1988, Biochem. Biophys. Res. Commun. 151: 25-33).

SEQ ID NO: 12 is Bacillus alkaline sp. K-38 amylase described as SEQ IDNO 2 in EP 1022334.

SEQ ID NO: 13 is a Bacillus licheniformis alpha-amylase described in Leeet al, 2006, J. Biochem, 139: 997-1005.

SEQ ID NO: 14 is a variant alpha-amylase LE399 previously disclosed in,e.g., WO 2002/010355.

DEFINITIONS

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-amylase: Alpha-amylases (E.C. 3.2.1.1) are a group of enzymeswhich catalyze the hydrolysis of starch and other linear and branched1,4 glucosidic oligo- and polysaccharides. The skilled person will knowhow to determine alpha-amylase activity. It may be determined accordingto the procedure described in the Examples, e.g., by the PNP-G7 assay.In one aspect, the variants of the present invention have at least 20%,e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, or at least 100% of the alpha-amylaseactivity of the mature polypeptide of SEQ ID NO: 1. In another aspect, avariant of the present application has at least 20%, e.g., at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or at least 100% of the alpha-amylase activity of its parent.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a variant. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding avariant of the present invention. Each control sequence may be native(i.e., from the same gene) or foreign (i.e., from a different gene) tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Expression: The term “expression” includes any step involved in theproduction of a variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantand is operably linked to control sequences that provide for itsexpression.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide; wherein the fragment has alpha-amylaseactivity. In one aspect, a fragment contains at least 300 amino acidresidues, at least 350 amino acid residues, at least 400 amino acidresidues, at least 450 amino acid residues, at least 470 amino acidresidues, or at least 480 amino acid residues.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentwhich does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. It is known in the art that a hostcell may produce a mixture of two or more different mature polypeptides(i.e., with a different C-terminal and/or N-terminal amino acid)expressed by the same polynucleotide. The mature form of somealpha-amylases, e.g., some bacterial alpha-amylases, comprises acatalytic domain containing the active site for substrate hydrolysis andone or more carbohydrate-binding modules (CBM) for binding to thecarbohydrate substrate (starch) and optionally a polypeptide linking theCBM(s) with the catalytic domain, a region of the latter type usuallybeing denoted a “linker”.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Parent or parent alpha-amylase: The term “parent” or “parentalpha-amylase” means an alpha-amylase to which an alteration is made toproduce the enzyme variants of the present invention. The parent may bea naturally occurring (wild-type) polypeptide or a variant or fragmentthereof.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the −nobrief option) is usedas the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

Variant: The term “variant” means a polypeptide having alpha-amylaseactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding one or more (e.g.,several) amino acids, e.g., 1-5 amino acids, adjacent to the amino acidoccupying a position. In one aspect, the variants of the presentinvention have at least 20%, e.g., at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least100% of the alpha-amylase activity of the mature polypeptide of SEQ IDNO: 1. In another aspect, a variant of the present application has atleast 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%,at least 80%, at least 90%, at least 95%, or at least 100% of thealpha-amylase activity of its parent. The alpha-amylase activity may bedetermined by the PNP-G7 assay described in the Examples.

Wild-type alpha-amylase: The term “wild-type” alpha-amylase means analpha-amylase expressed by a naturally occurring microorganism, such asa bacterium, yeast, or filamentous fungus found in nature.

CONVENTIONS FOR DESIGNATION OF VARIANTS

For purposes of the present invention, the mature polypeptide disclosedin SEQ ID NO: 1 is used to determine the corresponding amino acidresidue in another alpha-amylase. The amino acid sequence of anotheralpha-amylase is aligned with the mature polypeptide disclosed in SEQ IDNO: 1, and based on the alignment, the amino acid position numbercorresponding to any amino acid residue in the mature polypeptidedisclosed in SEQ ID NO: 1 is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends Genet. 16: 276-277), preferably version 5.0.0 or later. Theparameters used are gap open penalty of 10, gap extension penalty of0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

Identification of the corresponding amino acid residue in anotheralpha-amylase can be determined by an alignment of multiple polypeptidesequences using several computer programs including, but not limited to,MUSCLE (multiple sequence comparison by log-expectation; version 3.5 orlater; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT(version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518;Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009,Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010,Bioinformatics 26: 1899-1900), and EMBOSS EMMA employing ClustalW (1.83or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680),using their respective default parameters.

When the other enzyme has diverged from the mature polypeptide of SEQ IDNO: 1 such that traditional sequence-based comparison fails to detecttheir relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), other pairwise sequence comparison algorithms can be used.Greater sensitivity in sequence-based searching can be attained usingsearch programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Atschul etal., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivitycan be achieved if the family or superfamily for the polypeptide has oneor more representatives in the protein structure databases. Programssuch as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffinand Jones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letter amino acid abbreviation is employed.

Substitutions. For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, the substitution of threonine at position 226 withalanine is designated as “Thr226Ala” or “T226A”. Multiple mutations areseparated by addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or“G205R+S411F”, representing substitutions at positions 205 and 411 ofglycine (G) with arginine (R) and serine (S) with phenylalanine (F),respectively. In the Examples of the present application, multiplemutations are separated by a space, e.g., G205R S411F representingG205R+S411F.

Deletions. For an amino acid deletion, the following nomenclature isused: Original amino acid, position, *. Accordingly, the deletion ofglycine at position 195 is designated as “Gly195*” or “G195*”. Multipledeletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*”or “G195*+S411*”.

Insertions. For an amino acid insertion, the following nomenclature isused: Original amino acid, position, original amino acid, inserted aminoacid. Accordingly the insertion of lysine after glycine at position 195is designated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G - K - A

Multiple alterations. Variants comprising multiple alterations areseparated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or“R170Y+G195E” representing a substitution of arginine and glycine atpositions 170 and 195 with tyrosine and glutamic acid, respectively.

Different alterations. Where different alterations can be introduced ata position, the different alterations are separated by a comma, e.g.,“Arg170Tyr,Glu” represents a substitution of arginine at position 170with tyrosine or glutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala”designates the following variants:

“Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and“Tyr167Ala+Arg170Ala”.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to alpha-amylase variants comprising asubstitution with proline at a position corresponding to position 185 ofSEQ ID NO: 1 and further comprising a substitution at one or morepositions corresponding to positions 15, 48, 49, 50, 107, 116, 133, 138,156, 176, 181, 187, 188, 190, 197, 201, 205, 209, 213, 239, 241, 255,264, 299, 360, 375, 416, 437, 474 and 475 of SEQ ID NO: 1, wherein thevariant has at least 60% and less than 100% sequence identity to (i) themature polypeptide of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13 or 14, or (ii) amino acids 1 to 483 of SEQ ID NO: 1, aminoacids 1 to 483 of SEQ ID NO: 2, amino acids 1 to 485 of SEQ ID NO: 3,amino acids 1 to 482 of SEQ ID NO: 4, amino acids 1 to 484 of SEQ ID NO:5, amino acids 1 to 483 of SEQ ID NO: 6, amino acids 1 to 485 of SEQ IDNO: 7, amino acids 1 to 485 of SEQ ID NO: 8, amino acids 1 to 485 of SEQID NO: 9, amino acids 1 to 485 of SEQ ID NO: 10, amino acids 1 to 485 ofSEQ ID NO: 11, amino acids 1 to 480 of SEQ ID NO: 12, amino acids 1 to483 of SEQ ID NO: 13 or amino acids 1 to 481 of SEQ ID NO: 14, andwherein the variant has alpha-amylase activity.

Preferably, the variants are isolated.

Variants

The present invention provides alpha-amylase variants comprising asubstitution with proline at a position corresponding to position 185 ofSEQ ID NO: 1.

The variants further comprise a substitution at one or more (e.g.,several) positions corresponding to positions 15, 48, 49, 50, 107, 116,133, 138, 156, 176, 181, 187, 188, 190, 197, 201, 205, 209, 213, 239,241, 255, 264, 299, 360, 375, 416, 437, 474 and 475 of SEQ ID NO: 1.

The variants comprise a substitution at a position corresponding toposition 185 with Pro.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 15 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Leu, Ser or Thr.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 48 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Ala.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 49 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val, inparticular with Gly, His, Ile or Leu.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 50 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr or Val, inparticular with Thr.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 107 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Ala.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 116 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val, inparticular with Gly.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 133 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Tyr.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 138 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr or Val, inparticular with Phe or Tyr.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 156 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Tyr.

In one embodiment, the variant further comprises a substitution at aposition corresponding to position 176 with Ala, Arg, Asn, Asp, Cys,Gin, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val,in particular with Leu.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 181 with Arg, Asn, Asp, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Asp, Glu or Thr.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 187 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr or Val, inparticular with Asp.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 188 with Ala, Arg, Asp, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Ser or Thr.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 190 with Ala, Arg, Asp, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Phe.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 197 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Ile, Leu, Ser, Thr or Val.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 201 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Phe or Tyr.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 205 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Tyr.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 209 with Arg, Asn, Asp, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Val.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 213 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Thr.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 239 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr or Val, inparticular with Ala, Asn, Asp, Cys, Gin, Glu or Met.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 241 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Asp.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 255 with Ala, Arg, Asn, Asp, Cys, Gin, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Gly or Pro.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 264 with Ala, Arg, Asn, Asp, Cys, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Ser.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 299 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Arg.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 360 with Ala, Arg, Asn, Asp, Cys, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Ser.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 375 with Ala, Asn, Asp, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Gly or Val.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 416 with Ala, Arg, Asp, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Val.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 437 with Ala, Asn, Asp, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Trp.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 474 with Ala, Arg, Asn, Asp, Cys, Gin, Glu,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Arg, Gin, Glu or Lys.

In one embodiment, the variant comprises a substitution at a positioncorresponding to position 475 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, inparticular with Arg, Gln, Glu or Lys.

The variant has sequence identity of at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100%, to (i) the mature polypeptide of any of SEQ ID NOs: 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13 or 14, or (ii) amino acids 1 to 483 of SEQ IDNO: 1, amino acids 1 to 483 of SEQ ID NO: 2, amino acids 1 to 485 of SEQID NO: 3, amino acids 1 to 482 of SEQ ID NO: 4, amino acids 1 to 484 ofSEQ ID NO: 5, amino acids 1 to 483 of SEQ ID NO: 6, amino acids 1 to 485of SEQ ID NO: 7, amino acids 1 to 485 of SEQ ID NO: 8, amino acids 1 to485 of SEQ ID NO: 9, amino acids 1 to 485 of SEQ ID NO: 10, amino acids1 to 485 of SEQ ID NO: 11, amino acids 1 to 480 of SEQ ID NO: 12, aminoacids 1 to 483 of SEQ ID NO: 13 or amino acids 1 to 481 of SEQ ID NO:14.

In a preferred embodiment, the variant has at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99%, but lessthan 100%, sequence identity to (i) the mature polypeptide of SEQ ID NO:1, or (ii) amino acids 1-483 of SEQ ID NO: 1.

In one embodiment, the variant has at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 2,(ii) the mature polypeptide of SEQ ID NO: 2 comprising the deletions1181*+G182*, (iii) amino acids 1-483 of SEQ ID NO: 2, or (iv) aminoacids 1-483 of SEQ ID NO: 2 comprising the deletions I181*+G182*.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 3,(ii) the mature polypeptide of SEQ ID NO: 3 comprising the deletionsT183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 3, or (iv) aminoacids 1-485 of SEQ ID NO: 3 comprising the deletions T183*+G184*.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 4,(ii) the mature polypeptide of SEQ ID NO: 4 comprising the deletionsT180*+G181*, (iii) amino acids 1-482 of SEQ ID NO: 4, or (iv) aminoacids 1-482 of SEQ ID NO: 4 comprising the deletions T180*+G181*.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 5,(ii) the mature polypeptide of SEQ ID NO: 5 comprising the deletionsT182*+G183*, (iii) amino acids 1-484 of SEQ ID NO: 5, or (iv) aminoacids 1-484 of SEQ ID NO: 5 comprising the deletions T182*+G183*.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 6,(ii) the mature polypeptide of SEQ ID NO: 6 comprising the deletionsE178*+G179*, (iii) amino acids 1-483 of SEQ ID NO: 6, or (iv) aminoacids 1-483 of SEQ ID NO: 6 comprising the deletions E178*+G179*.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 7,(ii) the mature polypeptide of SEQ ID NO: 7 comprising the deletionsT183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 7, or (iv) aminoacids 1-485 of SEQ ID NO: 7 comprising the deletions T183*+G184*.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 8,(ii) the mature polypeptide of SEQ ID NO: 8 comprising the deletionsD183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 8, or (iv) aminoacids 1-485 of SEQ ID NO: 8 comprising the deletions D183*+G184*.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 9,(ii) the mature polypeptide of SEQ ID NO: 9 comprising the deletionsD183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 9, or (iv) aminoacids 1-485 of SEQ ID NO: 9 comprising the deletions D183*+G184*.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 10,(ii) the mature polypeptide of SEQ ID NO: 10 comprising the deletionsD183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 10, or (iv) aminoacids 1-485 of SEQ ID NO: 10 comprising the deletions D183*+G184*.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 11,(ii) the mature polypeptide of SEQ ID NO: 11 comprising the deletionsH183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 11, or (iv) aminoacids 1-485 of SEQ ID NO: 11 comprising the deletions H183*+G184*.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 12,or (ii) amino acids 1-480 of SEQ ID NO: 12.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99%, but less than100%, sequence identity to (i) the mature polypeptide of SEQ ID NO: 13,or (ii) amino acids 1-483 of SEQ ID NO: 13.

In another preferred embodiment, the variant has at least 60%, e.g., atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%, butless than 100%, sequence identity to (i) the mature polypeptide of SEQID NO: 14, or (ii) amino acids 1-481 of SEQ ID NO: 14.

A variant of the invention comprises a substitution with proline atposition 185. Further, the variant comprises a substitution at one ormore (e.g., several) positions corresponding to positions 15, 48, 49,50, 107, 116, 133, 138, 156, 176, 181, 187, 188, 190, 197, 201, 205,209, 213, 239, 241, 255, 264, 299, 360, 375, 416, 437, 474 and 475 ofSEQ ID NO: 1. In one embodiment, the variant comprises a substitution atone position corresponding to any of positions 15, 48, 49, 50, 107, 116,133, 138, 156, 176, 181, 187, 188, 190, 197, 201, 205, 209, 213, 239,241, 255, 264, 299, 360, 375, 416, 437, 474 and 475. In anotherembodiment, the variant comprises a substitution at two positionscorresponding to any of positions 15, 48, 49, 50, 107, 116, 133, 138,156, 176, 181, 187, 188, 190, 197, 201, 205, 209, 213, 239, 241, 255,264, 299, 360, 375, 416, 437, 474 and 475. In another embodiment, thevariant comprises a substitution at three positions corresponding to anyof positions 15, 48, 49, 50, 107, 116, 133, 138, 156, 176, 181, 187,188, 190, 197, 201, 205, 209, 213, 239, 241, 255, 264, 299, 360, 375,416, 437, 474 and 475. In another embodiment, the variant comprises asubstitution at four positions corresponding to positions 15, 48, 49,50, 107, 116, 133, 138, 156, 176, 181, 187, 188, 190, 197, 201, 205,209, 213, 239, 241, 255, 264, 299, 360, 375, 416, 437, 474 and 475. Inanother embodiment, the variant comprises a substitution at more thanfour, e.g., five, six, seven, eight, nine or ten, positionscorresponding to positions 15, 48, 49, 50, 107, 116, 133, 138, 156, 176,181, 187, 188, 190, 197, 201, 205, 209, 213, 239, 241, 255, 264, 299,360, 375, 416, 437, 474 and 475. In another embodiment, the variantcomprises a substitution at more than 10 positions, e.g., 11, 12, 13,14, 15, 16, 17, 18, 19 or 20 positions, corresponding to positions 15,48, 49, 50, 107, 116, 133, 138, 156, 176, 181, 187, 188, 190, 197, 201,205, 209, 213, 239, 241, 255, 264, 299, 360, 375, 416, 437, 474 and 475.

A variant of the invention comprises the substitution E185P. In oneembodiment, the variant further comprises one or more substitutionsselected from the group consisting of M15T, M15S, M15L, G48A, T49H,T49G, T49L, T49I, S50T, G107A, T116G, H133Y, W138Y, W138F, H156Y, K176L,A181T, A181E, A181D, S187D, N188S, N188T, N190F, M197S, M197T, M197V,M197L, M197I, I201Y, 1201F, H205Y, A209V, K213T, S239Q, S239E, S239N,S239D, S239A, S239M, S239C, L241D, E255P, E255G, Q264S, G299R, Q360S,R375V, R375G, D416V, R437W, G474K, G474R, G474E, G474Q, G475K, G475R,G475E and G475Q.

In a preferred embodiment, the variant comprises a substitution at oneor more positions corresponding to positions 49, 50, 116, 176, 201, 205,213, 241, 360, 375, 416, and 437. The variant preferably comprises oneor more substitutions selected among T49H, T49G, T49L, S50T, T116G,K176L, 1201Y, H205Y, K213T, L241D, Q360S, R375V, R375G, D416V and R437W.

In one embodiment, the variant comprises a substitution at one or morepositions corresponding to positions 49, 50, 116, 176, 201, 205, 213,241, 360, 375, 416, and 437 and further comprises a substitution at oneor more positions corresponding to positions 15, 48, 107, 133, 138, 156,181, 187, 188, 190, 197, 209, 239, 255, 264, 299, 474 and 475. Thevariant preferably comprises one or more substitutions selected amongT49H, T49G, T49L, S50T, T116G, K176L, 1201Y, H205Y, K213T, L241D, Q360S,R375V, R375G, D416V and R437W, and further one or more substitutionsselected among M15T, M15S, M15L, G48A, G107A, H133Y, W138Y, W138F,H156Y, A181T, A181E, A181D, S187D, N188S, N188T, N190F, M197S, M197T,M197V, M197L, M197I, A209V, S239Q, S239E, S239N, S239D, S239A, S239M,S239C, E255P, E255G, Q264S, G299R, G474K, G474R, G474E, G474Q, G475K,G475R, G475E and G475Q.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position15. Preferably, the variant comprises the substitutions M15T+E185P;M15S+E185P or M15L+E185P.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position48. Preferably, the variant comprises the substitutions G48A+E185P.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position49. Preferably, the variant comprises the substitutions T49H+E185P;T49G+E185P; T49L+E185P or T49I+E185P.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position50. Preferably, the variant comprises the substitutions S50T+E185P.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position107. Preferably, the variant comprises the substitutions G107A+E185P.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position116. Preferably, the variant comprises the substitutions T116G+E185P.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position133. Preferably, the variant comprises the substitutions H133Y+E185P.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position138. Preferably, the variant comprises the substitutions W138Y+E185P orW138F+E185P.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position156. Preferably, the variant comprises the substitutions H156Y+E185P.

In a preferred embodiment, the variant comprises the substitution E185Pin combination with a substitution at a position corresponding toposition 176. Preferably, the variant comprises the substitutionsK176L+E185P.

In another preferred embodiment, the variant comprises the substitutionE185P in combination with a substitution at a position corresponding toposition 176 and further in combination with a substitution at one ormore positions corresponding to positions 15, 48, 49, 50, 107, 116, 133,138, 156, 181, 187, 188, 190, 197, 201, 205, 209, 213, 239, 241, 255,264, 299, 360, 375, 416, 437, 474 and 475.

In another preferred embodiment, the variant comprises the substitutionsK176L+E185P and further comprises one or more substitutions selectedfrom the group consisting of M15T, M15S, M15L, G48A, T49H, T49G, T49L,T49I, S50T, G107A, T116G, H133Y, W138Y, W138F, H156Y, A181T, A181E,A181D, S187D, N188S, N188T, N190F, M197S, M197T, M197V, M197L, M197I,I201Y, 1201F, H205Y, A209V, K213T, S239Q, S239E, S239N, S239D, S239A,S239M, S239C, L241D, E255P, E255G, Q264S, G299R, Q360S, R375V, R375G,D416V, R437W, G474K, G474R, G474E, G474Q, G475K, G475R, G475E and G475Q.

In a more preferred embodiment, the variant comprises the substitutionsK176L+E185P and further comprises one or more substitutions selectedfrom the group consisting of T49H, T49G, T49L, S50T, T116G, 1201Y,H205Y, K213T, L241D, Q360S, R375V, R375G, D416V and R437W.

In another preferred embodiment, the variant comprises the substitutionE185P in combination with a substitution at a position corresponding toposition 176, wherein the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100%, sequence identity to SEQ ID NO: 1. Preferably, the variantcomprises the substitutions K176L+E185P, wherein the variant has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100%, sequence identity to SEQ ID NO: 1.

In another preferred embodiment, the variant comprises the substitutionE185P in combination with a substitution at a position corresponding toposition 176 and further in combination with a substitution at one ormore positions corresponding to positions 15, 48, 49, 50, 107, 116, 133,138, 156, 181, 187, 188, 190, 197, 201, 205, 209, 213, 239, 241, 255,264, 299, 360, 375, 416, 437, 474 and 475, wherein the variant has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100%, sequence identity to SEQ ID NO: 1.

In another preferred embodiment, the variant comprises the substitutionsK176L+E185P and further comprises one or more substitutions selectedfrom the group consisting of M15T, M15S, M15L, G48A, T49H, T49G, T49L,T49I, S50T, G107A, T116G, H133Y, W138Y, W138F, H156Y, A181T, A181E,A181D, S187D, N188S, N188T, N190F, M197S, M197T, M197V, M197L, M197I,I201Y, 1201F, H205Y, A209V, K213T, S239Q, S239E, S239N, S239D, S239A,S239M, S239C, L241D, E255P, E255G, Q264S, G299R, Q360S, R375V, R375G,D416V, R437W, G474K, G474R, G474E, G474Q, G475K, G475R, G475E and G475Q,wherein the variant has at least 60%, e.g., at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100%, sequenceidentity to SEQ ID NO: 1.

In a more preferred embodiment, the variant comprises the substitutionsK176L+E185P and further comprises one or more substitutions selectedfrom the group consisting of T49H, T49G, T49L, 550T, T116G, 1201Y,H205Y, K213T, L241D, Q360S, R375V, R375G, D416V and R437W, wherein thevariant has at least 60%, e.g., at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%, but less than 100%, sequenceidentity to SEQ ID NO: 1.

In another preferred embodiment, the variant comprises the substitutionE185P in combination with a substitution at a position corresponding toposition 176, wherein the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100%, sequence identity to SEQ ID NO: 14. Preferably, the variantcomprises the substitutions K176L+E185P, wherein the variant has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100%, sequence identity to SEQ ID NO: 14.

In another preferred embodiment, the variant comprises the substitutionE185P in combination with a substitution at a position corresponding toposition 176 and further in combination with a substitution at one ormore positions corresponding to positions 15, 48, 49, 50, 107, 116, 133,138, 156, 181, 187, 188, 190, 197, 201, 205, 209, 213, 239, 241, 255,264, 299, 360, 375, 416, 437, 474 and 475, wherein the variant has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100%, sequence identity to SEQ ID NO: 14.

In another preferred embodiment, the variant comprises the substitutionsK176L+E185P and further comprises one or more substitutions selectedfrom the group consisting of M15T, M15S, M15L, G48A, T49H, T49G, T49L,T49I, S50T, G107A, T116G, H133Y, W138Y, W138F, H156Y, A181T, A181E,A181D, S187D, N188S, N188T, N190F, M197S, M197T, M197V, M197L, M197I,1201Y, I201F, H205Y, A209V, K213T, S239Q, S239E, S239N, S239D, S239A,S239M, S239C, L241D, E255P, E255G, Q264S, G299R, Q360S, R375V, R375G,D416V, R437W, G474K, G474R, G474E, G474Q, G475K, G475R, G475E and G475Q,wherein the variant has at least 60%, e.g., at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100%, sequenceidentity to SEQ ID NO: 14.

In a more preferred embodiment, the variant comprises the substitutionsK176L+E185P and further comprises one or more substitutions selectedfrom the group consisting of T49H, T49G, T49L, 550T, T116G, 1201Y,H205Y, K213T, L241D, Q360S, R375V, R375G, D416V and R437W, wherein thevariant has at least 60%, e.g., at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%, but less than 100%, sequenceidentity to SEQ ID NO: 14.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position181. Preferably, the variant comprises the substitutions A181T+E185P;A181E+E185P or A181D+E185P.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position187. Preferably, the variant comprises the substitutions E185P+S187D.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position188. Preferably, the variant comprises the substitutions E185P+N188S orE185P+N188T.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position190. Preferably, the variant comprises the substitutions E185P+N190F.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position197. Preferably, the variant comprises the substitutions E185P+M197S,E185P+M197T, E185P+M197V, E185P+M197L or E185P+M197I.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position201. Preferably, the variant comprises the substitutions E185P+I201Y orE185P+I201F.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position205. Preferably, the variant comprises the substitutions E185P+H205Y.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position209. Preferably, the variant comprises the substitutions E185P+A209V.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position213. Preferably, the variant comprises the substitutions E185P+K213T.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position239. Preferably, the variant comprises the substitutions E185P+S239Q,E185P+S239E, E185P+S239N, E185P+S239D, E185P+S239A, E185P+S239M orE185P+S239C.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position241. Preferably, the variant comprises the substitutions E185P+L241D.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position255. Preferably, the variant comprises the substitutions E185P+E255P orE185P+E255G.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position264. Preferably, the variant comprises the substitutions E185P+Q264S.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position299. Preferably, the variant comprises the substitutions E185P+G299R.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position360. Preferably, the variant comprises the substitutions E185P+Q360S.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position375. Preferably, the variant comprises the substitutions E185P+R375V orE185P+R375G.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position416. Preferably, the variant comprises the substitutions E185P+D416V.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position437. Preferably, the variant comprises the substitutions E185P+R437W.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position474. Preferably, the variant comprises the substitutions E185P+474K,E185P+474R, E185P+474E or E185P+474Q.

In one embodiment, the variant comprises the substitution E185P incombination with a substitution at a position corresponding to position475. Preferably, the variant comprises the substitutions E185P+475K,E185P+475R, E185P+475E or E185P+475Q.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 15. Preferably, the variant comprises the substitutionsM15T+K176L+E185P; M15S+K176L+E185P or M15L+K176L+E185P.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 48. Preferably, the variant comprises the substitutionsG48A+K176L+E185P.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 49. Preferably, the variant comprises the substitutionsT49H+K176L+E185P; T49G+K176L+E185P; T49L+K176L+E185P orT49I+K176L+E185P.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 50. Preferably, the variant comprises the substitutionsS50T+K176L+E185P.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 107. Preferably, the variant comprises the substitutionsG107A+K176L+E185P.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 116. Preferably, the variant comprises the substitutionsT116G+K176L+E185P.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 133. Preferably, the variant comprises the substitutionsH133Y+K176L+E185P.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 138. Preferably, the variant comprises the substitutionsW138Y+K176L+E185P or W138F+K176L+E185P.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 156. Preferably, the variant comprises the substitutionsH156Y+K176L+E185P.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 181. Preferably, the variant comprises the substitutionsK176L+A181T+E185P; K176L+A181E+E185P or K176L+A181D+E185P.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 187. Preferably, the variant comprises the substitutionsK176L+E185P+S187D.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 188. Preferably, the variant comprises the substitutionsK176L+E185P+N188S or K176L+E185P+N188T.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 190. Preferably, the variant comprises the substitutionsK176L+E185P+N190F.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 197. Preferably, the variant comprises the substitutionsK176L+E185P+M197S, K176L+E185P+M197T, K176L+E185P+M197V,K176L+E185P+M197L or K176L+E185P+M197I.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 201. Preferably, the variant comprises the substitutionsK176L+E185P+1201Y or K176L+E185P+I201F.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 205. Preferably, the variant comprises the substitutionsK176L+E185P+H205Y.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 209. Preferably, the variant comprises the substitutionsK176L+E185P+A209V.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 213. Preferably, the variant comprises the substitutionsK176L+E185P+K213T.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 239. Preferably, the variant comprises the substitutionsK176L+E185P+S239Q, K176L+E185P+S239E, K176L+E185P+S239N,K176L+E185P+S239D, K176L+E185P+S239A, K176L+E185P+S239M orK176L+E185P+S239C.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 241. Preferably, the variant comprises the substitutionsK176L+E185P+L241D.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 255. Preferably, the variant comprises the substitutionsK176L+E185P+E255P or K176L+E185P+E255G.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 264. Preferably, the variant comprises the substitutionsK176L+E185P+Q264S.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 299. Preferably, the variant comprises the substitutionsK176L+E185P+G299R.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 360. Preferably, the variant comprises the substitutionsK176L+E185P+Q360S.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 375. Preferably, the variant comprises the substitutionsK176L+E185P+R375V or K176L+E185P+R375G.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 416. Preferably, the variant comprises the substitutionsK176L+E185P+D416V.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 437. Preferably, the variant comprises the substitutionsK176L+E185P+R437W.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 474. Preferably, the variant comprises the substitutionsK176L+E185P+G474K, K176L+E185P+G474R, K176L+E185P+G474E orK176L+E185P+G474Q.

In one embodiment, the variant comprises the substitutions E185P+K176Lin combination with a substitution at a position corresponding toposition 475. Preferably, the variant comprises the substitutionsK176L+E185P+G475K, K176L+E185P+G475R, K176L+E185P+G475E orK176L+E185P+G475Q.

In a preferred embodiment, the variant comprises a set of substitutionsselected from the group consisting of:

T49H+K176L+E185P,

T49G+K176L+E185P,

T49L+S50T+K176L+E185P,

T116G+K176L+E185P,

K176L+E185P,

K176L+E185P+1201Y+H205Y+K213T+Q360S+D416V+R437W,

K176L+E185P+L241D,

K176L+E185P+R375V, and

K176L+E185P+R375G.

In another preferred embodiment, the variant comprises a set ofsubstitutions selected from the group consisting of:

G48A+T49H+G107A+H156Y+K176L+A181T+E185P+N190F+1201F+A209V+Q264S;

G48A+T49G+G107A+H156Y+K176L+A181T+E185P+N190F+1201F+A209V+Q264S;

G48A+T49L+S50T+G107A+H156Y+K176L+A181T+E185P+N190F+1201F+A209V+Q264S;

G48A+T49I+G107A+T116G+H156Y+K176L+A181T+E185P+N190F+1201F+A209V+Q264S;

G48A+T49I+G107A+H156Y+K176L+A181T+E185P+N190F+1201F+A209V+Q264S;

G48A+T49I+G107A+H156Y+K176L+A181T+E185P+N190F+1201Y+H205Y+A209V+K213T+Q264S+Q360S+D416V+R437W;

G48A+T49I+G107A+H156Y+K176L+A181T+E185P+N190F+I201F+L241D+A209V+Q264S;

G48A+T49I+G107A+H156Y+K176L+A181T+E185P+N190F+1201F+A209V+Q264S+R375V;

G48A+T49I+G107A+H156Y+K176L+A181T+E185P+N190F+1201F+A209V+Q264S+R375G;and

G48A+G107A+H156Y+K176L+A181T+E185P+N190F+1201F+A209V+Q264S.

In one embodiment, the variant further comprises a deletion at both ofthe two positions immediately before the position corresponding toposition 180 of SEQ ID NO: 1. I.e., a deletion of the two amino acidscorresponding to positions 181 and 182 of SEQ ID NO: 2.

In another embodiment, the variant further comprises a deletion of twoamino acids after the position corresponding to position 177 of SEQ IDNO: 1 and before the position corresponding to position 180 of SEQ IDNO: 1. I.e., a deletion of two amino acids in the R179-G180-1181-G182peptide of SEQ ID NO: 2, or homologous amino acids in any of SEQ ID NOs:3 to 11.

The variants may further comprise one or more (e.g., several) additionalalterations, e.g., one or more (e.g., several) additional substitutions.

The additional amino acid changes may be of a minor nature, that isconservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of 1-30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to 20-25 residues; or a smallextension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for alpha-amylase activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide.

The variants may consist of 300 to 700, e.g., 350 to 650, 400 to 600,450 to 500 or 470 to 490, amino acids.

In a preferred embodiment, the variant has increased thermostabilitycompared to the mature polypeptide of SEQ ID NO: 1 at high temperature,low calcium and low pH.

In another preferred embodiment, the variant has increasedthermostability compared to the mature polypeptide of SEQ ID NO: 14 athigh temperature, low calcium and low pH.

In another preferred embodiment, the variant has increasedthermostability compared to its parent at high temperature, low calciumand low pH.

In the context of the present invention “high temperature” meanstemperatures from 70-120° C., preferably 80-100° C., more preferably85-95° C.

In the context of the present invention the term “low pH” means a pH inthe range from 4-6, preferably 4.2-5.5, more preferably 4.5-5.

In the context of the present invention the term “low calcium” meansfree calcium levels lower than 60 ppm, preferably 40 ppm, morepreferably 25 ppm, even more preferably 5 ppm calcium. 0.125 mM CaCl₂provides 5 ppm calcium.

In one embodiment, the variant has increased thermostability compared tothe mature polypeptide of SEQ ID NO: 1 when incubated with 0.125 mMCaCl₂ at 90° C. and pH 4.8. In another embodiment, the variant hasincreased thermostability compared to the mature polypeptide of SEQ IDNO: 1 when incubated with 0.125 mM CaCl₂ at 95° C. and pH 4.8. Inanother embodiment, the variant has increased thermostability comparedto the mature polypeptide of SEQ ID NO: 1 when incubated with 0.125 mMCaCl₂ at 90° C. and pH 4.5. In another embodiment, the variant hasincreased thermostability compared to the mature polypeptide of SEQ IDNO: 1 when incubated with 0.125 mM CaCl₂) at 95° C. and pH 4.5.

In one embodiment, the variant has increased thermostability compared tothe mature polypeptide of SEQ ID NO: 14 when incubated with 0.125 mMCaCl₂) at 90° C. and pH 4.8. In another embodiment, the variant hasincreased thermostability compared to the mature polypeptide of SEQ IDNO: 14 when incubated with 0.125 mM CaCl₂) at 95° C. and pH 4.8. Inanother embodiment, the variant has increased thermostability comparedto the mature polypeptide of SEQ ID NO: 14 when incubated with 0.125 mMCaCl₂) at 90° C. and pH 4.5. In another embodiment, the variant hasincreased thermostability compared to the mature polypeptide of SEQ IDNO: 14 when incubated with 0.125 mM CaCl₂) at 95° C. and pH 4.5.

In one embodiment, the variant has increased thermostability compared toits parent when incubated with 0.125 mM CaCl₂ at 90° C. and pH 4.8. Inanother embodiment, the variant has increased thermostability comparedto its parent when incubated with 0.125 mM CaCl₂ at 95° C. and pH 4.8.In another embodiment, the variant has increased thermostabilitycompared to its parent when incubated with 0.125 mM CaCl₂ at 90° C. andpH 4.5. In another embodiment, the variant has increased thermostabilitycompared to its parent when incubated with 0.125 mM CaCl₂ at 95° C. andpH 4.5.

The skilled person will know how to determine thermostability of theenzymes. It may be done by determining the residual activity half-lifeas shown in the Examples of the present application. The skilled personwill know how to choose the relevant conditions for the assay, such asthe incubation time.

In a preferred embodiment, the variant has an increased residualactivity half-life compared to the mature polypeptide of SEQ ID NO: 1 athigh temperature, low calcium and low pH. In one embodiment, the varianthas an increased residual activity half-life compared to the maturepolypeptide of SEQ ID NO: 1 when incubated with 0.125 mM CaCl₂) at 90°C. and pH 4.8. In another embodiment, the variant has an increasedresidual activity half-life compared to the mature polypeptide of SEQ IDNO: 1 when incubated with 0.125 mM CaCl₂ at 95° C. and pH 4.8. Inanother embodiment, the variant has an increased residual activityhalf-life compared to the mature polypeptide of SEQ ID NO: 1 whenincubated with 0.125 mM CaCl₂ at 90° C. and pH 4.5. In anotherembodiment, the variant has an increased residual activity half-lifecompared to the mature polypeptide of SEQ ID NO: 1 when incubated with0.125 mM CaCl₂ at 95° C. and pH 4.5.

In a preferred embodiment, the variant has an increased residualactivity half-life compared to the mature polypeptide of SEQ ID NO: 14at high temperature, low calcium and low pH. In one embodiment, thevariant has an increased residual activity half-life compared to themature polypeptide of SEQ ID NO: 14 when incubated with 0.125 mM CaCl₂at 90° C. and pH 4.8. In another embodiment, the variant has anincreased residual activity half-life compared to the mature polypeptideof SEQ ID NO: 14 when incubated with 0.125 mM CaCl₂) at 95° C. and pH4.8. In another embodiment, the variant has an increased residualactivity half-life compared to the mature polypeptide of SEQ ID NO: 14when incubated with 0.125 mM CaCl₂) at 90° C. and pH 4.5. In anotherembodiment, the variant has an increased residual activity half-lifecompared to the mature polypeptide of SEQ ID NO: 14 when incubated with0.125 mM CaCl₂) at 95° C. and pH 4.5.

In a preferred embodiment, the variant has a residual activityhalf-life, when incubated with 0.125 mM CaCl₂) at 90° C. and pH 4.8,which is at least 5 minutes, e.g., at least 10 minutes or at least 20minutes. In a more preferred embodiment, the variant has a residualactivity half-life, when incubated with 0.125 mM CaCl₂ at 90° C. and pH4.8, which is at least 50 minutes, e.g., at least 60 minutes or at least70 minutes. In one embodiment, the variant has a residual activityhalf-life, when incubated with 0.125 mM CaCl₂ at 90° C. and pH 4.8,which is at least 100 minutes.

In a preferred embodiment, the variant has a residual activityhalf-life, when incubated with 0.125 mM CaCl₂ at 90° C. and pH 4.5,which is at least 2 minute, e.g., at least 3 minutes or at least 5minutes. In a more preferred embodiment, the variant has a residualactivity half-life, when incubated with 0.125 mM CaCl₂ at 90° C. and pH4.5, which is at least 10 minutes, e.g., at least 15 minutes or at least20 minutes. In one embodiment, the variant has a residual activityhalf-life, when incubated with 0.125 mM CaCl₂) at 90° C. and pH 4.5,which is at least 30 minutes.

In a preferred embodiment, the variant has a residual activityhalf-life, when incubated with 0.125 mM CaCl₂) at 95° C. and pH 4.8,which is at least 1 minute, e.g., at least 2 minutes or at least 5minutes. In a more preferred embodiment, the variant has a residualactivity half-life, when incubated with 0.125 mM CaCl₂) at 95° C. and pH4.8, which is at least 10 minutes, e.g., at least 15 minutes or at least20 minutes. In one embodiment, the variant has a residual activityhalf-life, when incubated with 0.125 mM CaCl₂ at 95° C. and pH 4.8,which is at least 30 minutes.

In a preferred embodiment, the variant has a residual activityhalf-life, when incubated with 0.125 mM CaCl₂ at 95° C. and pH 4.5,which is at least 1 minute, e.g., at least 2 minutes or at least 3minutes. In a more preferred embodiment, the variant has a residualactivity half-life, when incubated with 0.125 mM CaCl₂ at 95° C. and pH4.5, which is at least 5 minutes, e.g., at least 7 minutes or at least10 minutes. In one embodiment, the variant has a residual activityhalf-life, when incubated with 0.125 mM CaCl₂ at 95° C. and pH 4.8,which is at least 15 minutes.

In one embodiment, the variant has increased thermostability, e.g., anincreased residual activity half-life, compared to the parent enzymewhen incubated with 0.125 mM CaCl₂ at 90° C. and pH 4.8. In anotherembodiment, the variant has increased thermostability, e.g., anincreased residual activity half-life, compared to the parent enzymewhen incubated with 0.125 mM CaCl₂) at 95° C. and pH 4.8. In anotherembodiment, the variant has increased thermostability, e.g., anincreased residual activity half-life, compared to the parent enzymewhen incubated with 0.125 mM CaCl₂) at 90° C. and pH 4.5. In anotherembodiment, the variant has increased thermostability, e.g., anincreased residual activity half-life, compared to the parent enzymewhen incubated with 0.125 mM CaCl₂) at 95° C. and pH 4.5.

In other embodiments, the variant has improved catalytic efficiency,improved catalytic rate, improved chemical stability, improved oxidationstability, improved specific activity, improved stability under storageconditions, improved substrate binding, improved substrate cleavage,improved substrate specificity, improved substrate stability, improvedsurface properties, improved thermal activity, or improvedthermostability compared to the parent enzyme.

Parent Alpha-Amylase

The variant is preferably a variant of a parent alpha-amylase selectedfrom the group consisting of:

(a) a polypeptide having at least 60% sequence identity to (i) themature polypeptide of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13 or 14, or (ii) amino acids 1 to 483 of SEQ ID NO: 1, aminoacids 1 to 483 of SEQ ID NO: 2, amino acids 1 to 485 of SEQ ID NO: 3,amino acids 1 to 482 of SEQ ID NO: 4, amino acids 1 to 484 of SEQ ID NO:5, amino acids 1 to 483 of SEQ ID NO: 6, amino acids 1 to 485 of SEQ IDNO: 7, amino acids 1 to 485 of SEQ ID NO: 8, amino acids 1 to 485 of SEQID NO: 9, amino acids 1 to 485 of SEQ ID NO: 10, amino acids 1 to 485 ofSEQ ID NO: 11, amino acids 1 to 480 of SEQ ID NO: 12, amino acids 1 to483 of SEQ ID NO: 13 or amino acids 1 to 481 of SEQ ID NO: 14; or

(b) a fragment of the mature polypeptide of any of SEQ ID NOs: 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, which has alpha-amylaseactivity.

In one embodiment, the parent alpha-amylase has at least 65%, e.g., atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of any of SEQ IDNOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or (ii) aminoacids 1 to 483 of SEQ ID NO: 1, amino acids 1 to 483 of SEQ ID NO: 2,amino acids 1 to 485 of SEQ ID NO: 3, amino acids 1 to 482 of SEQ ID NO:4, amino acids 1 to 484 of SEQ ID NO: 5, amino acids 1 to 483 of SEQ IDNO: 6, amino acids 1 to 485 of SEQ ID NO: 7, amino acids 1 to 485 of SEQID NO: 8, amino acids 1 to 485 of SEQ ID NO: 9, amino acids 1 to 485 ofSEQ ID NO: 10, amino acids 1 to 485 of SEQ ID NO: 11, amino acids 1 to480 of SEQ ID NO: 12, amino acids 1 to 483 of SEQ ID NO: 13 or aminoacids 1 to 481 of SEQ ID NO: 14.

In one embodiment, the parent alpha-amylase has at least 60%, e.g., atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to (i) the mature polypeptide of SEQID NO: 1, or (ii) amino acids 1-483 of SEQ ID NO: 1.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 2,(ii) the mature polypeptide of SEQ ID NO: 2 comprising the deletionsI181*+G182*, (iii) amino acids 1-483 of SEQ ID NO: 2, or (iv) aminoacids 1-483 of SEQ ID NO: 2 comprising the deletions I181*+G182*.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 3,(ii) the mature polypeptide of SEQ ID NO: 3 comprising the deletionsT183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 3, or (iv) aminoacids 1-485 of SEQ ID NO: 3 comprising the deletions T183*+G184*.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 4,(ii) the mature polypeptide of SEQ ID NO: 4 comprising the deletionsT180*+G181*, (iii) amino acids 1-482 of SEQ ID NO: 4, or (iv) aminoacids 1-482 of SEQ ID NO: 4 comprising the deletions T180*+G181*.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 5,(ii) the mature polypeptide of SEQ ID NO: 5 comprising the deletionsT182*+G183*, (iii) amino acids 1-484 of SEQ ID NO: 5, or (iv) aminoacids 1-484 of SEQ ID NO: 5 comprising the deletions T182*+G183*.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 6,(ii) the mature polypeptide of SEQ ID NO: 6 comprising the deletionsE178*+G179*, (iii) amino acids 1-483 of SEQ ID NO: 6, or (iv) aminoacids 1-483 of SEQ ID NO: 6 comprising the deletions E178*+G179*.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 7,(ii) the mature polypeptide of SEQ ID NO: 7 comprising the deletionsT183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 7, or (iv) aminoacids 1-485 of SEQ ID NO: 7 comprising the deletions T183*+G184*.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 8,(ii) the mature polypeptide of SEQ ID NO: 8 comprising the deletionsD183*+G184*, (iii) amino acids 1-486 of SEQ ID NO: 8, or (iv) aminoacids 1-486 of SEQ ID NO: 8 comprising the deletions D183*+G184*.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 9,(ii) the mature polypeptide of SEQ ID NO: 9 comprising the deletionsD183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 9, or (iv) aminoacids 1-485 of SEQ ID NO: 9 comprising the deletions D183*+G184*.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 10,(ii) the mature polypeptide of SEQ ID NO: 10 comprising the deletionsD183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 10, or (iv) aminoacids 1-485 of SEQ ID NO: 10 comprising the deletions D183*+G184*.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 11,(ii) the mature polypeptide of SEQ ID NO: 11 comprising the deletionsH183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 11, or (iv) aminoacids 1-485 of SEQ ID NO: 11 comprising the deletions H183*+G184*.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 12,or (ii) amino acids 1-480 of SEQ ID NO: 12.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 13,or (ii) amino acids 1-483 of SEQ ID NO: 13.

In another embodiment, the parent alpha-amylase has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to (i) the mature polypeptide of SEQ ID NO: 14,or (ii) amino acids 1-481 of SEQ ID NO: 14.

In one embodiment, the parent alpha-amylase comprises or consists of themature polypeptide of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13 or 14.

In one embodiment, the parent alpha-amylase comprises or consists ofamino acids 1 to 483 of SEQ ID NO: 1, amino acids 1 to 483 of SEQ ID NO:2, amino acids 1 to 485 of SEQ ID NO: 3, amino acids 1 to 482 of SEQ IDNO: 4, amino acids 1 to 484 of SEQ ID NO: 5, amino acids 1 to 483 of SEQID NO: 6, amino acids 1 to 485 of SEQ ID NO: 7, amino acids 1 to 485 ofSEQ ID NO: 8, amino acids 1 to 485 of SEQ ID NO: 9, amino acids 1 to 485of SEQ ID NO: 10, amino acids 1 to 485 of SEQ ID NO: 11, amino acids 1to 480 of SEQ ID NO: 12, amino acids 1 to 483 of SEQ ID NO: 13 or aminoacids 1 to 481 of SEQ ID NO: 14.

In another embodiment, the parent alpha-amylase is a fragment of themature polypeptide of any of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13 or 14, wherein the fragment has alpha-amylase activity.

In one embodiment, the variant has at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, suchas at least 96%, at least 97%, at least 98%, or at least 99%, but lessthan 100%, sequence identity to the parent alpha-amylase.

In one embodiment, the total number of alterations in the variants ofthe present invention is 2-20, e.g., 2-10 and 2-5, such as 2, 3, 4, 5,6, 7, 8, 9 or 10 alterations.

The parent may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The parent may be a fusion polypeptide or cleavable fusion polypeptidein which another polypeptide is fused at the N-terminus or theC-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

The parent may be obtained from microorganisms of any genus. Forpurposes of the present invention, the term “obtained from” as usedherein in connection with a given source shall mean that the parentencoded by a polynucleotide is produced by the source or by a strain inwhich the polynucleotide from the source has been inserted. In oneaspect, the parent is secreted extracellularly.

The parent may be a bacterial alpha-amylase. For example, the parent maybe a Gram-positive bacterial polypeptide such as a Bacillus,Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus,Oceanobacillus, Staphylococcus, Streptococcus, or Streptomycesalpha-amylase, or a Gram-negative bacterial polypeptide such as aCampylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasmaalpha-amylase.

In one aspect, the parent is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis alpha-amylase.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms and DNA directly from natural habitats are wellknown in the art. A polynucleotide encoding a parent may then beobtained by similarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding aparent has been detected with the probe(s), the polynucleotide can beisolated or cloned by utilizing techniques that are known to those ofordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Preparation of Variants

The present invention also relates to methods for obtaining a varianthaving alpha-amylase activity, comprising: (a) introducing into a parentalpha-amylase a substitution with proline at a position corresponding toposition 185 of SEQ ID NO: 1 and further a substitution at one or morepositions corresponding to positions 15, 48, 49, 50, 107, 116, 133, 138,156, 176, 181, 187, 188, 190, 197, 201, 205, 209, 213, 239, 241, 255,264, 299, 360, 375, 416, 437, 474 and 475 of SEQ ID NO: 1; and (b)recovering the variant.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga variant of the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide which isrecognized by a host cell for expression of the polynucleotide. Thepromoter contains transcriptional control sequences that mediate theexpression of the variant. The promoter may be any polynucleotide thatshows transcriptional activity in the host cell including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from an Aspergillus neutral alpha-amylasegene in which the untranslated leader has been replaced by anuntranslated leader from an Aspergillus triose phosphate isomerase gene;non-limiting examples include modified promoters from an Aspergillusniger neutral alpha-amylase gene in which the untranslated leader hasbeen replaced by an untranslated leader from an Aspergillus nidulans orAspergillus oryzae triose phosphate isomerase gene); and mutant,truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminatorsequence is operably linked to the 3′-terminus of the polynucleotideencoding the variant. Any terminator that is functional in the host cellmay be used.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leadersequence is operably linked to the 5′-terminus of the polynucleotideencoding the variant. Any leader that is functional in the host cell maybe used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase,

Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiaealcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase(ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the variant-encoding sequence and,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding sequence naturally linked in translation reading framewith the segment of the coding sequence that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding sequence that is foreign to the coding sequence. Aforeign signal peptide coding sequence may be required where the codingsequence does not naturally contain a signal peptide coding sequence.Alternatively, a foreign signal peptide coding sequence may simplyreplace the natural signal peptide coding sequence in order to enhancesecretion of the variant. However, any signal peptide coding sequencethat directs the expressed variant into the secretory pathway of a hostcell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a variant. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of the variantand the signal peptide sequence is positioned next to the N-terminus ofthe propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the variant relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the variant would be operably linkedwith the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promoter, and transcriptional and translational stop signals. Thevarious nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the variant at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAM111permitting replication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the production of avariant of the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thevariant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see,e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see,e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any methodknown in the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phiebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a variant,comprising: (a) cultivating a host cell of the present invention underconditions suitable for expression of the variant; and (b) recoveringthe variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the variantto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant can be recovered directly from the medium. If the variant is notsecreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that arespecific for the variants. For example, an enzyme assay may be used todetermine the activity of the variant.

The variant may be recovered using methods known in the art. Forexample, the variant may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The variant may be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure variants.

In an alternative aspect, the variant is not recovered, but rather ahost cell of the present invention expressing the variant is used as asource of the variant.

Compositions

The present invention also relates to compositions comprising analpha-amylase variant and at least one additional enzyme. The additionalenzyme(s) may be selected from the group consisting of beta-amylase,cellulase (beta-glucosidase, cellobiohydrolase and endoglucanase),glucoamylase, hemicellulsae (e.g., xylanase), isoamylase, isomerase,lipase, phytase, protease, pullulanase, and/or other enzymes useful in acommercial process in conjunction with an alpha-amylase. The additionalenzyme may also be a second alpha-amylase. Such enzymes are known in theart in starch processing, sugar conversion, fermentations for alcoholand other useful end-products, commercial detergents and cleaning aids,stain removal, fabric treatment or desizing, and the like.

Uses

The variants of the present invention possess valuable propertiesallowing for a variety of industrial applications. In particular, thevariants may be used in detergents, in particular laundry detergentcompositions and dishwashing detergent compositions, hard surfacecleaning compositions, and for desizing textiles, fabrics or garments,production of pulp and paper, beer making, ethanol production, andstarch conversion processes.

The alpha-amylase variants may be used for starch processes, inparticular starch conversion, especially liquefaction of starch (see,e.g., U.S. Pat. No. 3,912,590, EP 252730 and EP 063909, WO 99/19467, andWO 96/28567, which are all hereby incorporated by reference). Alsocontemplated are compositions for starch conversion purposes, which maybeside the variant of the invention also comprise an AMG, pullulanase,and other alpha-amylases.

Further, the variants are particularly useful in the production ofsweeteners and ethanol (see, e.g., U.S. Pat. No. 5,231,017, which ishereby incorporated by reference), such as fuel, drinking and industrialethanol, from starch or whole grains.

The variants may also be used for desizing of textiles, fabrics, andgarments (see, e.g., WO 95/21247, U.S. Pat. No. 4,643,736, and EP119920, which are incorporated herein by reference), beer making orbrewing, and in pulp and paper production or related processes.

Starch Processing

Native starch consists of microscopic granules, which are insoluble inwater at room temperature. When an aqueous starch slurry is heated, thegranules swell and eventually burst, dispersing the starch moleculesinto the solution. During this “gelatinization” process there is adramatic increase in viscosity. As the solids level is 30-40% in atypical industrial process, the starch has to be thinned or “liquefied”so that it can be suitably processed. This reduction in viscosity isprimarily attained by enzymatic degradation in current commercialpractice.

Conventional starch-conversion processes, such as liquefaction andsaccharification processes are described, e.g., in U.S. Pat. No.3,912,590, EP 252730 and EP 063909, which are incorporated herein byreference.

In an embodiment, the conversion process degrading starch to lowermolecular weight carbohydrate components such as sugars or fat replacersincludes a debranching step.

In the case of converting starch into a sugar, the starch isdepolymerized. Such a depolymerization process consists of, e.g., apre-treatment step and two or three consecutive process steps, i.e., aliquefaction process, a saccharification process, and depending on thedesired end-product, an optional isomerization process.

When the desired final sugar product is, e.g., high fructose syrup thedextrose syrup may be converted into fructose. After thesaccharification process, the pH is increased to a value in the range of6-8, e.g., pH 7.5, and the calcium is removed by ion exchange. Thedextrose syrup is then converted into high fructose syrup using, e.g.,an immobilized glucose isomerase.

Production of Fermentation Products

In general, alcohol production (ethanol) from whole grain can beseparated into 4 main steps: milling, liquefaction, saccharification,and fermentation.

The grain is milled in order to open up the structure and allow forfurther processing. Two processes used are wet or dry milling. In drymilling, the whole kernel is milled and used in the remaining part ofthe process. Wet milling gives a very good separation of germ and meal(starch granules and protein) and is with a few exceptions applied atlocations where there is a parallel production of syrups.

In the liquefaction process the starch granules are solubilized byhydrolysis to maltodextrins mostly of a DP higher than 4. The hydrolysismay be carried out by acid treatment or enzymatically by analpha-amylase. Acid hydrolysis is used on a limited basis. The rawmaterial can be milled whole grain or a side stream from starchprocessing.

During a typical enzymatic liquefaction, the long-chained starch isdegraded into branched and linear shorter units (maltodextrins) by analpha-amylase. Enzymatic liquefaction is generally carried out as athree-step hot slurry process. The slurry is heated to between 60-95° C.(e.g., 77-86° C., 80-85° C., or 83-85° C.) and the enzyme(s) is (are)added. The liquefaction process is carried out at 85° C. for 1-2 hours.The pH is generally between 5.5 and 6.2. In order to ensure optimalenzyme stability under these conditions, 1 mM of calcium is added (toprovide about 40 ppm free calcium ions). After such treatment, theliquefied starch will have a “dextrose equivalent” (DE) of 10-15.

The slurry is subsequently jet-cooked at between 95-140° C., e.g.,105-125° C., cooled to 60-95° C. and more enzyme(s) is (are) added toobtain the final hydrolysis. The liquefaction process is carried out atpH 4.5-6.5, typically at a pH between 5 and 6. Milled and liquefiedgrain is also known as mash.

Liquefied starch-containing material is saccharified in the presence ofsaccharifying enzymes such as glucoamylases. The saccharificationprocess may last for 12 hours to 120 hours (e.g., 12 to 90 hours, 12 to60 hours and 12 to 48 hours).

However, it is common to perform a pre-saccharification step for about30 minutes to 2 hours (e.g., 30 to 90 minutes) at a temperature of 30 to65° C., typically around 60° C. which is followed by a completesaccharification during fermentation referred to as simultaneoussaccharification and fermentation (SSF). The pH is usually between4.2-4.8, e.g., pH 4.5. In a simultaneous saccharification andfermentation (SSF) process, there is no holding stage forsaccharification, rather, the yeast and enzymes are added together.

In a typical saccharification process, maltodextrins produced duringliquefaction are converted into dextrose by adding a glucoamylase and adebranching enzyme, such as an isoamylase (U.S. Pat. No. 4,335,208) or apullulanase. The temperature is lowered to 60° C., prior to the additionof the glucoamylase and debranching enzyme. The saccharification processproceeds for 24-72 hours.

Prior to addition of the saccharifying enzymes, the pH is reduced tobelow 4.5, while maintaining a high temperature (above 95° C.), toinactivate the liquefying alpha-amylase. This process reduces theformation of short oligosaccharide called “panose precursors,” whichcannot be hydrolyzed properly by the debranching enzyme. Normally, about0.2-0.5% of the saccharification product is the branched trisaccharidepanose (Glc pα1-6Glc pα1-4Glc), which cannot be degraded by apullulanase. If active amylase from the liquefaction remains presentduring saccharification (i.e., no denaturing), the amount of panose canbe as high as 1-2%, which is highly undesirable since it lowers thesaccharification yield significantly.

Fermentable sugars (e.g., dextrins, monosaccharides, particularlyglucose) are produced from enzymatic saccharification. These fermentablesugars may be further purified and/or converted to useful sugarproducts. In addition, the sugars may be used as a fermentationfeedstock in a microbial fermentation process for producingend-products, such as alcohol (e.g., ethanol and butanol), organic acids(e.g., succinic acid and lactic acid), sugar alcohols (e.g., glycerol),ascorbic acid intermediates (e.g., gluconate, 2-keto-D-gluconate,2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid), amino acids (e.g.,lysine), proteins (e.g., antibodies and fragment thereof).

In an embodiment, the fermentable sugars obtained during theliquefaction process steps are used to produce alcohol and particularlyethanol. In ethanol production, an SSF process is commonly used whereinthe saccharifying enzymes and fermenting organisms (e.g., yeast) areadded together and then carried out at a temperature of 30-40° C.

The organism used in fermentation will depend on the desiredend-product. Typically, if ethanol is the desired end product yeast willbe used as the fermenting organism. In some preferred embodiments, theethanol-producing microorganism is a yeast and specificallySaccharomyces such as strains of S. cerevisiae (U.S. Pat. No.4,316,956). A variety of S. cerevisiae are commercially available andthese include but are not limited to FALI (Fleischmann's Yeast),SUPERSTART (Alltech), FERMIOL (DSM Specialties), RED STAR (Lesaffre) andAngel alcohol yeast (Angel Yeast Company, China). The amount of starteryeast employed in the methods is an amount effective to produce acommercially significant amount of ethanol in a suitable amount of time,(e.g., to produce at least 10% ethanol from a substrate having between25-40% DS in less than 72 hours). Yeast cells are generally supplied inamounts of about 10⁴ to about 10¹², and preferably from about 10⁷ toabout 10¹⁰ viable yeast count per mL of fermentation broth. After yeastis added to the mash, it is typically subjected to fermentation forabout 24-96 hours, e.g., 35-60 hours. The temperature is between about26-34° C., typically at about 32° C., and the pH is from pH 3-6, e.g.,around pH 4-5.

The fermentation may include, in addition to a fermenting microorganisms(e.g., yeast), nutrients, and additional enzymes, including phytases.The use of yeast in fermentation is well known in the art.

In further embodiments, use of appropriate fermenting microorganisms, asis known in the art, can result in fermentation end product including,e.g., glycerol, 1,3-propanediol, gluconate, 2-keto-D-gluconate,2,5-diketo-D-gluconate, 2-keto-L-gulonic acid, succinic acid, lacticacid, amino acids, and derivatives thereof. More specifically whenlactic acid is the desired end product, a Lactobacillus sp. (L. casei)may be used; when glycerol or 1,3-propanediol are the desiredend-products E. coli may be used; and when 2-keto-D-gluconate,2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid are the desired endproducts, Pantoea citrea may be used as the fermenting microorganism.The above enumerated list is only examples and one skilled in the artwill be aware of a number of fermenting microorganisms that may be usedto obtain a desired end product.

Processes for Producing Fermentation Products from UngelatinizedStarch-Containing Material

The invention relates to processes for producing fermentation productsfrom starch-containing material without gelatinization (i.e., withoutcooking) of the starch-containing material. The fermentation product,such as ethanol, can be produced without liquefying the aqueous slurrycontaining the starch-containing material and water. In one embodiment aprocess of the invention includes saccharifying (e.g., milled)starch-containing material, e.g., granular starch, below the initialgelatinization temperature, preferably in the presence of alpha-amylaseand/or carbohydrate-source generating enzyme(s) to produce sugars thatcan be fermented into the fermentation product by a suitable fermentingorganism. In this embodiment the desired fermentation product, e.g.,ethanol, is produced from ungelatinized (i.e., uncooked), preferablymilled, cereal grains, such as corn. Accordingly, in the first aspectthe invention relates to processes for producing fermentation productsfrom starch-containing material comprising simultaneously saccharifyingand fermenting starch-containing material using a carbohydrate-sourcegenerating enzyme and a fermenting organism at a temperature below theinitial gelatinization temperature of said starch-containing material.In an embodiment a protease is also present. The protease may be anyacid fungal protease or metalloprotease. The fermentation product, e.g.,ethanol, may optionally be recovered after fermentation, e.g., bydistillation. Typically amylase(s), such as glucoamylase(s) and/or othercarbohydrate-source generating enzymes, and/or alpha-amylase(s), is(are)present during fermentation. Examples of glucoamylases and othercarbohydrate-source generating enzymes include raw starch hydrolyzingglucoamylases. Examples of alpha-amylase(s) include acid alpha-amylasessuch as acid fungal alpha-amylases. Examples of fermenting organismsinclude yeast, e.g., a strain of Saccharomyces cerevisiae. The term“initial gelatinization temperature” means the lowest temperature atwhich starch gelatinization commences. In general, starch heated inwater begins to gelatinize between about 50° C. and 75° C.; the exacttemperature of gelatinization depends on the specific starch and canreadily be determined by the skilled artisan. Thus, the initialgelatinization temperature may vary according to the plant species, tothe particular variety of the plant species as well as with the growthconditions. In the context of this invention the initial gelatinizationtemperature of a given starch-containing material may be determined asthe temperature at which birefringence is lost in 5% of the starchgranules using the method described by Gorinstein and Lii, 1992,Starch/Stärke 44(12): 461-466. Before initiating the process a slurry ofstarch-containing material, such as granular starch, having 10-55 w/w %dry solids (DS), preferably 25-45 w/w % dry solids, more preferably30-40 w/w % dry solids of starch-containing material may be prepared.The slurry may include water and/or process waters, such as stillage(backset), scrubber water, evaporator condensate or distillate,side-stripper water from distillation, or process water from otherfermentation product plants. Because the process of the invention iscarried out below the initial gelatinization temperature, and thus nosignificant viscosity increase takes place, high levels of stillage maybe used if desired. In an embodiment the aqueous slurry contains fromabout 1 to about 70 vol. %, preferably 15-60 vol. %, especially fromabout 30 to 50 vol. % water and/or process waters, such as stillage(backset), scrubber water, evaporator condensate or distillate,side-stripper water from distillation, or process water from otherfermentation product plants, or combinations thereof, or the like. Thestarch-containing material may be prepared by reducing the particlesize, preferably by dry or wet milling, to 0.05 to 3.0 mm, preferably0.1-0.5 mm. After being subjected to a process of the invention at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or preferably at least99% of the dry solids in the starch-containing material are convertedinto a soluble starch hydrolyzate. A process in this aspect of theinvention is conducted at a temperature below the initial gelatinizationtemperature, which means that the temperature typically lies in therange between 30-75° C., preferably between 45-60° C. In a preferredembodiment the process carried at a temperature from 25° C. to 40° C.,such as from 28° C. to 35° C., such as from 30° C. to 34° C., preferablyaround 32° C. In an embodiment the process is carried out so that thesugar level, such as glucose level, is kept at a low level, such asbelow 6 w/w %, such as below about 3 w/w %, such as below about 2 w/w %,such as below about 1 w/w %, such as below about 0.5 w/w %, or below0.25 w/w %, such as below about 0.1 w/w %. Such low levels of sugar canbe accomplished by simply employing adjusted quantities of enzyme andfermenting organism. A skilled person in the art can easily determinewhich doses/quantities of enzyme and fermenting organism to use. Theemployed quantities of enzyme and fermenting organism may also beselected to maintain low concentrations of maltose in the fermentationbroth. For instance, the maltose level may be kept below about 0.5 w/w%, such as below about 0.2 w/w %. The process of the invention may becarried out at a pH from about 3 and 7, preferably from pH 3.5 to 6, ormore preferably from pH 4 to 5. In an embodiment fermentation is ongoingfor 6 to 120 hours, in particular 24 to 96 hours.

Processes for Producing Fermentation Products from GelatinizedStarch-Containing Material

In this aspect the invention relates to processes for producingfermentation products, especially ethanol, from starch-containingmaterial, which process includes a liquefaction step and sequentially orsimultaneously performed saccharification and fermentation steps.Consequently, the invention relates to processes for producingfermentation products from starch-containing material comprising thesteps of:

(a) liquefying starch-containing material in the presence of analpha-amylase variant, or

(b) saccharifying the liquefied material obtained in step (a) using acarbohydrate-source generating enzyme;

(c) fermenting using a fermenting organism.

In an aspect, a pullulanase such as a family GH57 pullulanase is alsoused in the liquefaction step. In an embodiment a protease, such as anacid fungal protease or a metallo protease is added before, duringand/or after liquefaction. In an embodiment the metalloprotease isderived from a strain of Thermoascus, e.g., a strain of Thermoascusaurantiacus, especially Thermoascus aurantiacus CGMCC No. 0670. In anembodiment the carbohydrate-source generating enzyme is a glucoamylasederived from a strain of Aspergillus, e.g., Aspergillus niger orAspergillus awamori, a strain of Talaromyces, especially Talaromycesemersonii; or a strain of Athelia, especially Athelia rolfsii; a strainof Trametes, e.g., Trametes cingulata; a strain of the genusPachykytospora, e.g., a strain of Pachykytospora papyracea; or a strainof the genus Leucopaxillus, e.g., Leucopaxillus giganteus; or a strainof the genus Peniophora, e.g., a strain of the species Peniophorarufomarginata; or a mixture thereof. Saccharification step (b) andfermentation step (c) may be carried out either sequentially orsimultaneously. A pullulanase and/or metalloprotease may be added duringsaccharification and/or fermentation when the process is carried out asa sequential saccharification and fermentation process and before orduring fermentation when steps (b) and (c) are carried outsimultaneously (SSF process). The pullulanase and/or metalloprotease mayalso advantageously be added before liquefaction (pre-liquefactiontreatment), i.e., before or during step (a), and/or after liquefaction(post liquefaction treatment), i.e., after step (a). The pullulanase ismost advantageously added before or during liquefaction, i.e., before orduring step (a). The fermentation product, such as especially ethanol,may optionally be recovered after fermentation, e.g., by distillation.The fermenting organism is preferably yeast, preferably a strain ofSaccharomyces cerevisiae. In a particular embodiment, the process of theinvention further comprises, prior to step (a), the steps of:

x) reducing the particle size of the starch-containing material,preferably by milling (e.g., using a hammer mill);

y) forming a slurry comprising the starch-containing material and water.

In an embodiment the particle size is smaller than a #7 screen, e.g., a#6 screen. A #7 screen is usually used in conventional prior artprocesses. The aqueous slurry may contain from 10-55, e.g., 25-45 and30-40, w/w % dry solids (DS) of starch-containing material. The slurryis heated to above the gelatinization temperature and an alpha-amylasevariant may be added to initiate liquefaction (thinning). The slurry mayin an embodiment be jet-cooked to further gelatinize the slurry beforebeing subjected to alpha-amylase in step (a). Liquefaction may in anembodiment be carried out as a three-step hot slurry process. The slurryis heated to between 60-95° C., preferably between 70-90° C., such aspreferably between 80-85° C. at pH 4-6, preferably 4.5-5.5, andalpha-amylase variant, optionally together with a pullulanase and/orprotease, preferably metalloprotease, are added to initiate liquefaction(thinning). In an embodiment the slurry may then be jet-cooked at atemperature between 95-140° C., preferably 100-135° C., such as 105-125°C., for about 1-15 minutes, preferably for about 3-10 minutes,especially around about 5 minutes. The slurry is cooled to 60-95° C. andmore alpha-amylase variant and optionally pullulanase variant and/orprotease, preferably metalloprotease, is(are) added to finalizehydrolysis (secondary liquefaction). The liquefaction process is usuallycarried out at pH 4.0-6, in particular at a pH from 4.5 to 5.5.Saccharification step (b) may be carried out using conditions well knownin the art. For instance, a full saccharification process may last up tofrom about 24 to about 72 hours, however, it is common only to do apre-saccharification of typically 40-90 minutes at a temperature between30-65° C., typically about 60° C., followed by complete saccharificationduring fermentation in a simultaneous saccharification and fermentationprocess (SSF process). Saccharification is typically carried out attemperatures from 20-75° C., preferably from 40-70° C., typically around60° C., and at a pH between 4 and 5, normally at about pH 4.5. The mostwidely used process to produce a fermentation product, especiallyethanol, is a simultaneous saccharification and fermentation (SSF)process, in which there is no holding stage for the saccharification,meaning that a fermenting organism, such as yeast, and enzyme(s), may beadded together. SSF may typically be carried out at a temperature from25° C. to 40° C., such as from 28° C. to 35° C., such as from 30° C. to34° C., preferably around about 32° C. In an embodiment fermentation isongoing for 6 to 120 hours, in particular 24 to 96 hours.

Beer Making

The alpha-amylase variants may also be used in a beer-making process andsimilar fermentations; the alpha-amylases will typically be added duringthe mashing process. The process is substantially similar to themilling, liquefaction, saccharification, and fermentation processesdescribed above.

Starch Slurry Processing with Stillage

Milled starch-containing material is combined with water and recycledthin-stillage resulting in an aqueous slurry. The slurry can comprisebetween 15 to 55% DS w/w (e.g., 20 to 50%, 25 to 50%, 25 to 45%, 25 to40%, 20 to 35% and 30-36% DS). In some embodiments, the recycledthin-stillage (backset) is in the range of about 10 to 70% v/v (e.g., 10to 60%, 10 to 50%, 10 to 40%, 10 to 30%, 10 to 20%, 20 to 60%, 20 to50%, 20 to 40% and also 20 to 30%).

Once the milled starch-containing material is combined with water andbackset, the pH is not adjusted in the slurry. Further the pH is notadjusted after the addition of a phytase and optionally an alpha-amylaseto the slurry. In an embodiment, the pH of the slurry will be in therange of about pH 4.5 to less than about 6.0 (e.g., pH 4.5 to 5.8, pH4.5 to 5.6, pH 4.8 to 5.8, pH 5.0 to 5.8, pH 5.0 to 5.4, pH 5.2 to 5.5and pH 5.2 to 5.9). The pH of the slurry may be between about pH 4.5 and5.2 depending on the amount of thin stillage added to the slurry and thetype of material comprising the thin stillage. For example, the pH ofthe thin stillage may be between pH 3.8 and pH 4.5.

During ethanol production, acids can be added to lower the pH in thebeer well, to reduce the risk of microbial contamination prior todistillation.

In some embodiments, a phytase is added to the slurry. In otherembodiments, in addition to phytase, an alpha-amylase is added to theslurry. In some embodiments, a phytase and alpha-amylase are added tothe slurry sequentially. In other embodiments, a phytase andalpha-amylase are added simultaneously. In some embodiments, the slurrycomprising a phytase and optionally, an alpha-amylase, are incubated(pretreated) for a period of about 5 minutes to about 8 hours (e.g., 5minutes to 6 hours, 5 minutes to 4 hours, 5 minutes to 2 hours, and 15minutes to 4 hours). In other embodiments, the slurry is incubated at atemperature in the range of about 40 to 115° C. (e.g., 45 to 80° C., 50to 70° C., 50 to 75° C., 60 to 110° C., 60 to 95° C., 70 to 110° C., 70to 85° C. and 77 to 86° C.).

In other embodiments, the slurry is incubated at a temperature of about0 to about 30° C. (e.g., 0 to 25° C., 0 to 20° C., 0 to 15° C., 0 to 10°C. and 0 to 5° C.) below the starch gelatinization temperature of thestarch-containing material. In some embodiments, the temperature isbelow about 68° C., below about 65° C., below about 62° C., below about60° C. and below about 55° C. In some embodiments, the temperature isabove about 45° C., above about 50° C., above about 55° C. and aboveabout 60° C. In some embodiments, the incubation of the slurrycomprising a phytase and an alpha-amylase at a temperature below thestarch gelatinization temperature is referred to as a primary (1°)liquefaction.

In one embodiment, the milled starch-containing material is corn ormilo. The slurry comprises 25 to 40% DS, the pH is in the range of 4.8to 5.2, and the slurry is incubated with a phytase and optionally analpha-amylase for 5 minutes to 2 hours, at a temperature range of 60 to75° C.

Currently, it is believed that commercially available microbialalpha-amylases used in the liquefaction process are generally not stableenough to produce liquefied starch substrate from a dry mill processusing whole ground grain at a temperature above about 80° C. at a pHlevel that is less than pH 5.6. The stability of many commerciallyavailable alpha-amylases is reduced at low pH.

In a further liquefaction step, the incubated or pretreatedstarch-containing material is exposed to an increase in temperature suchas about 0 to about 45° C. above the starch gelatinization temperatureof the starch-containing material (e.g., 70° C. to 120° C., 70° C. to110° C., and 70° C. to 90° C.) for a period of time of about 2 minutesto about 6 hours (e.g., 2 minutes to 4 hrs, 90 minutes, 140 minutes and90 to 140 minutes) at a pH of about 4.0 to 5.5 more preferably between 1hour to 2 hours. The temperature can be increased by a conventional hightemperature jet cooking system for a short period of time, for example,for 1 to 15 minutes. Then the starch may be further hydrolyzed at atemperature ranging from about 75° C. to 95° C. (e.g., 80° C. to 90° C.and 80° C. to 85° C.) for a period of about 15 to 150 minutes (e.g., 30to 120 minutes). In a preferred embodiment, the pH is not adjustedduring these process steps and the pH of the liquefied mash is in therange of about pH 4.0 to pH 5.8 (e.g., pH 4.5 to 5.8, pH 4.8 to 5.4, andpH 5.0 to 5.2). In some embodiments, a second dose of thermostablealpha-amylase is added to the secondary liquefaction step, but in otherembodiments there is no additional dosage of alpha-amylase.

The incubation and liquefaction steps may be followed bysaccharification and fermentation steps well known in the art.

Distillation

Optionally, following fermentation, an alcohol (e.g., ethanol) may beextracted by, for example, distillation and optionally followed by oneor more process steps.

In some embodiments, the yield of ethanol produced by the methodsprovided herein is at least 8%, at least 10%, at least 12%, at least14%, at least 15%, at least 16%, at least 17% and at least 18% (v/v) andat least 23% v/v. The ethanol obtained according to the process providedherein may be used as, for example, fuel ethanol, drinking ethanol,i.e., potable neutral spirits, or industrial ethanol.

By-Products

Left over from the fermentation is the grain, which is typically usedfor animal feed either in liquid or dried form. In further embodiments,the end product may include the fermentation co-products such asdistiller's dried grains (DDG) and distiller's dried grain plus solubles(DDGS), which may be used, for example, as an animal feed.

Further details on how to carry out liquefaction, saccharification,fermentation, distillation, and recovery of ethanol are well known tothe skilled person.

According to the process provided herein, the saccharification andfermentation may be carried out simultaneously or separately.

Pulp and Paper Production

The alpha-amylase variants may also be used in the production oflignocellulosic materials, such as pulp, paper and cardboard, fromstarch reinforced waste paper and cardboard, especially where re-pulpingoccurs at pH above 7 and where amylases facilitate the disintegration ofthe waste material through degradation of the reinforcing starch. Thealpha-amylase variants are especially useful in a process for producinga papermaking pulp from starch-coated printed-paper. The process may beperformed as described in WO 95/14807, comprising the following steps:

a) disintegrating the paper to produce a pulp,

b) treating with a starch-degrading enzyme before, during or after stepa), and

c) separating ink particles from the pulp after steps a) and b).

The alpha-amylase variants may also be useful in modifying starch whereenzymatically modified starch is used in papermaking together withalkaline fillers such as calcium carbonate, kaolin and clays. With thealpha-amylase variants it is possible to modify the starch in thepresence of the filler thus allowing for a simpler integrated process.

Desizing of Textiles, Fabrics and Garments

The alpha-amylase variants may also be very useful in textile, fabric orgarment desizing. In the textile processing industry, alpha-amylases aretraditionally used as auxiliaries in the desizing process to facilitatethe removal of starch-containing size, which has served as a protectivecoating on weft yarns during weaving. Complete removal of the sizecoating after weaving is important to ensure optimum results in thesubsequent processes, in which the fabric is scoured, bleached and dyed.Enzymatic starch breakdown is preferred because it does not involve anyharmful effect on the fiber material. In order to reduce processing costand increase mill throughput, the desizing process is sometimes combinedwith the scouring and bleaching steps. In such cases, non-enzymaticauxiliaries such as alkali or oxidation agents are typically used tobreak down the starch, because traditional alpha-amylases are not verycompatible with high pH levels and bleaching agents. The non-enzymaticbreakdown of the starch size leads to some fiber damage because of therather aggressive chemicals used. Accordingly, it would be desirable touse the alpha-amylase variants as they have an improved performance inalkaline solutions. The alpha-amylase variants may be used alone or incombination with a cellulase when desizing cellulose-containing fabricor textile.

Desizing and bleaching processes are well known in the art. Forinstance, such processes are described in e.g., WO 95/21247, U.S. Pat.No. 4,643,736, EP 119920, which are hereby incorporated by reference.

Cleaning Processes and Detergent Compositions

The alpha-amylase variants may be added as a component of a detergentcomposition for various cleaning or washing processes, including laundryand dishwashing. For example, the variants may be used in the detergentcompositions described in WO 96/23874 and WO 97/07202.

The alpha-amylase variants may be incorporated in detergents atconventionally employed concentrations. For example, a variant of theinvention may be incorporated in an amount corresponding to 0.00001-10mg (calculated as pure, active enzyme protein) of alpha-amylase perliter of wash/dishwash liquor using conventional dosing levels ofdetergent.

The detergent composition may for example be formulated as a hand ormachine laundry detergent composition, including a laundry additivecomposition suitable for pretreatment of stained fabrics and a rinseadded fabric softener composition or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dishwashing operations.

The detergent composition may further comprise one or more otherenzymes, such as a lipase, peroxidase, protease, another amylolyticenzyme, e.g., another alpha-amylase, glucoamylase, maltogenic amylase,CGTase, cellulase, mannanase (such as Mannaway™ from Novozymes,Denmark)), pectinase, pectin lyase, cutinase, and/or laccase.

In general the properties of the chosen enzyme(s) should be compatiblewith the selected detergent (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additive,e.g., a separate additive or a combined additive, can be formulated,e.g., granulate, a liquid, a slurry, etc. Preferred detergent additiveformulations are granulates, in particular non-dusting granulates,liquids, in particular stabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonyl-phenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols, fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238216.

The detergent composition may be in any convenient form, e.g., a bar, atablet, a powder, a granule, a paste or a liquid. A liquid detergent maybe aqueous, typically containing up to about 70% water and 0 to about30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of fromabout 0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonyl-phenol ethoxylate, alkylpolyglycoside, alkyldimethylamine-oxide,ethoxylated fatty acid monoethanol-amide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0 to about 65% of a detergent builder orcomplexing agent such as zeolite, diphosphate, triphosphate,phosphonate, carbonate, citrate, nitrilotriacetic acid,ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid,alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates(e.g., SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinyl-pyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleiclacrylic acid copolymersand lauryl methacrylate/acrylic acid co-polymers.

The detergent may contain a bleaching system, which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxyben-zenesul-fonate. Alternatively, the bleaching system maycomprise peroxy acids of, e.g., the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition may be stabilized usingconventional stabilizing agents, e.g., a polyol such as propylene glycolor glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or aboric acid derivative, e.g., an aromatic borate ester, or a phenylboronic acid derivative such as 4-formylphenyl boronic acid, and thecomposition may be formulated as described in, e.g., WO 92/19708 and WO92/19709.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilre-deposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

The detergent compositions may comprise any enzyme in an amountcorresponding to 0.01-100 mg of enzyme protein per liter of wash liquor,preferably 0.055 mg of enzyme protein per liter of wash liquor, inparticular 0.1-1 mg of enzyme protein per liter of wash liquor.

One or more of the variant enzymes described herein may additionally beincorporated in the detergent formulations disclosed in WO 97/07202,which is hereby incorporated as reference.

The invention is further defined by the following embodiments:

Embodiment 1

An alpha-amylase variant comprising a substitution with proline at aposition corresponding to position 185 of SEQ ID NO: 1 and furthercomprising a substitution at one or more positions corresponding topositions 15, 48, 49, 50, 107, 116, 133, 138, 156, 176, 181, 187, 188,190, 197, 201, 205, 209, 213, 239, 241, 255, 264, 299, 360, 375, 416,437, 474 and 475 of SEQ ID NO: 1, wherein the variant has at least 60%and less than 100% sequence identity to (i) the mature polypeptide ofany of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or(ii) amino acids 1 to 483 of SEQ ID NO: 1, amino acids 1 to 483 of SEQID NO: 2, amino acids 1 to 485 of SEQ ID NO: 3, amino acids 1 to 482 ofSEQ ID NO: 4, amino acids 1 to 484 of SEQ ID NO: 5, amino acids 1 to 483of SEQ ID NO: 6, amino acids 1 to 485 of SEQ ID NO: 7, amino acids 1 to485 of SEQ ID NO: 8, amino acids 1 to 485 of SEQ ID NO: 9, amino acids 1to 485 of SEQ ID NO: 10, amino acids 1 to 485 of SEQ ID NO: 11, aminoacids 1 to 480 of SEQ ID NO: 12, amino acids 1 to 483 of SEQ ID NO: 13or amino acids 1 to 481 of SEQ ID NO: 14, and wherein the variant hasalpha-amylase activity.

Embodiment 2

The variant of Embodiment 1, which comprises:

a substitution at a position corresponding to position 15 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr,Trp, Tyr or Val, in particular with Leu, Ser or Thr;

a substitution at a position corresponding to position 48 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr or Val, in particular with Ala;

a substitution at a position corresponding to position 49 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Trp, Tyr or Val, in particular with Gly, His, Ile or Leu;

a substitution at a position corresponding to position 50 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr,Trp, Tyr or Val, in particular with Thr;

a substitution at a position corresponding to position 107 with Ala,Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val, in particular with Ala;

a substitution at a position corresponding to position 116 with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Trp, Tyr or Val, in particular with Gly;

a substitution at a position corresponding to position 133 with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val, in particular with Tyr;

a substitution at a position corresponding to position 138 with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Tyr or Val, in particular with Phe or Tyr;

a substitution at a position corresponding to position 156 with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val, in particular with Tyr;

a substitution at a position corresponding to position 176 with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val, in particular with Leu;

a substitution at a position corresponding to position 181 with Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val, in particular with Asp, Glu or Thr;

a substitution at a position corresponding to position 187 with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Thr, Trp, Tyr or Val, in particular with Asp;

a substitution at a position corresponding to position 188 with Ala,Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val, in particular with Ser or Thr;

a substitution at a position corresponding to position 190 with Ala,Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val, in particular with Phe;

a substitution at a position corresponding to position 197 with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser,Thr, Trp, Tyr or Val, in particular with Ile, Leu, Ser, Thr or Val;

a substitution at a position corresponding to position 201 with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val, in particular with Phe or Tyr;

a substitution at a position corresponding to position 205 with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val, in particular with Tyr;

a substitution at a position corresponding to position 209 with Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val, in particular with Val;

a substitution at a position corresponding to position 213 with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val, in particular with Thr;

a substitution at a position corresponding to position 239 with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Thr, Trp, Tyr or Val, in particular with Ala, Asn, Asp, Cys, Gln, Glu orMet;

a substitution at a position corresponding to position 241 with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val, in particular with Asp;

a substitution at a position corresponding to position 255 with Ala,Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val, in particular with Gly or Pro;

a substitution at a position corresponding to position 264 with Ala,Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val, in particular with Ser;

a substitution at a position corresponding to position 299 with Ala,Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val, in particular with Arg;

a substitution at a position corresponding to position 360 with Ala,Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val, in particular with Ser;

a substitution at a position corresponding to position 375 with Ala,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val, in particular with Gly or Val;

a substitution at a position corresponding to position 416 with Ala,Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val, in particular with Val;

a substitution at a position corresponding to position 437 with Ala,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val, in particular with Trp;

a substitution at a position corresponding to position 474 with Ala,Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val, in particular with Arg, Gln, Glu or Lys; or

a substitution at a position corresponding to position 475 with Ala,Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,Thr, Trp, Tyr or Val, in particular with Arg, Gln, Glu or Lys.

Embodiment 3

The variant of Embodiment 1 or 2, wherein the variant has at least 65%,e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98% or at least 99%, but lessthan 100%, sequence identity to (i) the mature polypeptide of any of SEQID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or (ii) aminoacids 1 to 483 of SEQ ID NO: 1, amino acids 1 to 483 of SEQ ID NO: 2,amino acids 1 to 485 of SEQ ID NO: 3, amino acids 1 to 482 of SEQ ID NO:4, amino acids 1 to 484 of SEQ ID NO: 5, amino acids 1 to 483 of SEQ IDNO: 6, amino acids 1 to 485 of SEQ ID NO: 7, amino acids 1 to 485 of SEQID NO: 8, amino acids 1 to 485 of SEQ ID NO: 9, amino acids 1 to 485 ofSEQ ID NO: 10, amino acids 1 to 485 of SEQ ID NO: 11, amino acids 1 to480 of SEQ ID NO: 12, amino acids 1 to 483 of SEQ ID NO: 13 or aminoacids 1 to 481 of SEQ ID NO: 14.

Embodiment 4

The variant of Embodiment 1 or 2, wherein the variant has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99%, but less than 100%, sequence identity to (i) the mature polypeptideof SEQ ID NO: 1, or (ii) amino acids 1-483 of SEQ ID NO: 1.

Embodiment 5

The variant of Embodiment 1 or 2, wherein the variant has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99%, but less than 100%, sequence identity to (i) the mature polypeptideof SEQ ID NO: 2, (ii) the mature polypeptide of SEQ ID NO: 2 comprisingthe deletions I181*+G182*, (iii) amino acids 1-483 of SEQ ID NO: 2, or(iv) amino acids 1-483 of SEQ ID NO: 2 comprising the deletionsI181*+G182*.

Embodiment 6

The variant of Embodiment 1 or 2, wherein the variant has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99%, but less than 100%, sequence identity to (i) the mature polypeptideof SEQ ID NO: 3, (ii) the mature polypeptide of SEQ ID NO: 3 comprisingthe deletions T183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 3, or(iv) amino acids 1-485 of SEQ ID NO: 3 comprising the deletionsT183*+G184*.

Embodiment 7

The variant of Embodiment 1 or 2, wherein the variant has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% sequence identity, but less than 100%, to (i) the mature polypeptideof SEQ ID NO: 4, (ii) the mature polypeptide of SEQ ID NO: 4 comprisingthe deletions T180*+G181*, (iii) amino acids 1-482 of SEQ ID NO: 4, or(iv) amino acids 1-482 of SEQ ID NO: 4 comprising the deletionsT180*+G181*.

Embodiment 8

The variant of Embodiment 1 or 2, wherein the variant has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99%, but less than 100%, sequence identity to (i) the mature polypeptideof SEQ ID NO: 5, (ii) the mature polypeptide of SEQ ID NO: 5 comprisingthe deletions T182*+G183*, (iii) amino acids 1-484 of SEQ ID NO: 5, or(iv) amino acids 1-484 of SEQ ID NO: 5 comprising the deletionsT182*+G183*.

Embodiment 9

The variant of Embodiment 1 or 2, wherein the variant has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99%, but less than 100%, sequence identity to (i) the mature polypeptideof SEQ ID NO: 6, (ii) the mature polypeptide of SEQ ID NO: 6 comprisingthe deletions E178*+G179*, (iii) amino acids 1-483 of SEQ ID NO: 6, or(iv) amino acids 1-483 of SEQ ID NO: 6 comprising the deletionsE178*+G180*.

Embodiment 10

The variant of Embodiment 1 or 2, wherein the variant has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99%, but less than 100%, sequence identity to (i) the mature polypeptideof SEQ ID NO: 7, (ii) the mature polypeptide of SEQ ID NO: 7 comprisingthe deletions T183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 7, or(iv) amino acids 1-485 of SEQ ID NO: 7 comprising the deletionsT183*+G184*.

Embodiment 11

The variant of Embodiment 1 or 2, wherein the variant has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99%, but less than 100%, sequence identity to (i) the mature polypeptideof SEQ ID NO: 8, (ii) the mature polypeptide of SEQ ID NO: 8 comprisingthe deletions D183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 8, or(iv) amino acids 1-485 of SEQ ID NO: 8 comprising the deletionsD183*+G184*.

Embodiment 12

The variant of Embodiment 1 or 2, wherein the variant has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99%, but less than 100%, sequence identity to (i) the mature polypeptideof SEQ ID NO: 9, (ii) the mature polypeptide of SEQ ID NO: 9 comprisingthe deletions D183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 9, or(iv) amino acids 1-485 of SEQ ID NO: 9 comprising the deletionsD183*+G184*.

Embodiment 13

The variant of Embodiment 1 or 2, wherein the variant has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99%, but less than 100%, sequence identity to (i) the mature polypeptideof SEQ ID NO: 10, (ii) the mature polypeptide of SEQ ID NO: 10comprising the deletions D183*+G184*, (iii) amino acids 1-485 of SEQ IDNO: 10, or (iv) amino acids 1-485 of SEQ ID NO: 10 comprising thedeletions D183*+G184*.

Embodiment 14

The variant of Embodiment 1 or 2, wherein the variant has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99%, but less than 100%, sequence identity to (i) the mature polypeptideof SEQ ID NO: 11, (ii) the mature polypeptide of SEQ ID NO: 11comprising the deletions H183*+G184*, (iii) amino acids 1-485 of SEQ IDNO: 11, or (iv) amino acids 1-485 of SEQ ID NO: 11 comprising thedeletions H183*+G184*.

Embodiment 15

The variant of Embodiment 1 or 2, wherein the variant has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99%, but less than 100%, sequence identity to (i) the mature polypeptideof SEQ ID NO: 12, or (ii) amino acids 1-480 of SEQ ID NO: 12.

Embodiment 16

The variant of Embodiment 1 or 2, wherein the variant has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99%, but less than 100%, sequence identity to (i) the mature polypeptideof SEQ ID NO: 13, or (ii) amino acids 1-483 of SEQ ID NO: 13.

Embodiment 17

The variant of Embodiment 1 or 2, wherein the variant has at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% or at least99%, but less than 100%, sequence identity to (i) the mature polypeptideof SEQ ID NO: 14, or (ii) amino acids 1-481 of SEQ ID NO: 14.

Embodiment 18

The variant of any of Embodiments 1-17, which is a variant of a parentalpha-amylase selected from the group consisting of:

(a) a polypeptide having at least 60% sequence identity to (i) themature polypeptide of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13 or 14, or (ii) amino acids 1 to 483 of SEQ ID NO: 1, aminoacids 1 to 483 of SEQ ID NO: 2, amino acids 1 to 485 of SEQ ID NO: 3,amino acids 1 to 482 of SEQ ID NO: 4, amino acids 1 to 484 of SEQ ID NO:5, amino acids 1 to 483 of SEQ ID NO: 6, amino acids 1 to 485 of SEQ IDNO: 7, amino acids 1 to 485 of SEQ ID NO: 8, amino acids 1 to 485 of SEQID NO: 9, amino acids 1 to 485 of SEQ ID NO: 10, amino acids 1 to 485 ofSEQ ID NO: 11, amino acids 1 to 480 of SEQ ID NO: 12, amino acids 1 to483 of SEQ ID NO: 13 or amino acids 1 to 481 of SEQ ID NO: 14; or

(b) a fragment of the mature polypeptide of any of SEQ ID NOs: 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, which has alpha-amylaseactivity.

Embodiment 19

The variant of Embodiment 18, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to (i) the maturepolypeptide of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13 or 14, or (ii) amino acids 1 to 483 of SEQ ID NO: 1, amino acids 1 to483 of SEQ ID NO: 2, amino acids 1 to 485 of SEQ ID NO: 3, amino acids 1to 482 of SEQ ID NO: 4, amino acids 1 to 484 of SEQ ID NO: 5, aminoacids 1 to 483 of SEQ ID NO: 6, amino acids 1 to 485 of SEQ ID NO: 7,amino acids 1 to 485 of SEQ ID NO: 8, amino acids 1 to 485 of SEQ ID NO:9, amino acids 1 to 485 of SEQ ID NO: 10, amino acids 1 to 485 of SEQ IDNO: 11, amino acids 1 to 480 of SEQ ID NO: 12, amino acids 1 to 483 ofSEQ ID NO: 13 or amino acids 1 to 481 of SEQ ID NO: 14.

Embodiment 20

The variant of Embodiment 18, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to (i) the maturepolypeptide of SEQ ID NO: 1, or (ii) amino acids 1-483 of SEQ ID NO: 1.

Embodiment 21

The variant of Embodiment 18, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to (i) the maturepolypeptide of SEQ ID NO: 2, (ii) the mature polypeptide of SEQ ID NO: 2comprising the deletions I181*+G182*, (iii) amino acids 1-483 of SEQ IDNO: 2, or (iv) amino acids 1-483 of SEQ ID NO: 2 comprising thedeletions I181*+G182*.

Embodiment 22

The variant of Embodiment 18, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to (i) the maturepolypeptide of SEQ ID NO: 3, (ii) the mature polypeptide of SEQ ID NO: 3comprising the deletions T183*+G184*, (iii) amino acids 1-485 of SEQ IDNO: 3, or (iv) amino acids 1-485 of SEQ ID NO: 3 comprising thedeletions T183*+G184*.

Embodiment 23

The variant of Embodiment 18, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to (i) the maturepolypeptide of SEQ ID NO: 4, (ii) the mature polypeptide of SEQ ID NO: 4comprising the deletions T180*+G181*, (iii) amino acids 1-482 of SEQ IDNO: 4, or (iv) amino acids 1-482 of SEQ ID NO: 4 comprising thedeletions T180*+G181*.

Embodiment 24

The variant of Embodiment 18, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to (i) the mature polypeptide of SEQID NO: 5, (ii) the mature polypeptide of SEQ ID NO: 5 comprising thedeletions T182*+G183*, (iii) amino acids 1-484 of SEQ ID NO: 5, or (iv)amino acids 1-484 of SEQ ID NO: 5 comprising the deletions T182*+G183*.

Embodiment 25

The variant of Embodiment 18, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to (i) the mature polypeptide of SEQID NO: 6, (ii) the mature polypeptide of SEQ ID NO: 6 comprising thedeletions E178*+G179*, (iii) amino acids 1-483 of SEQ ID NO: 6, or (iv)amino acids 1-483 of SEQ ID NO: 6 comprising the deletions E178*+G179*.

Embodiment 26

The variant of Embodiment 18, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to (i) the mature polypeptide of SEQID NO: 7, (ii) the mature polypeptide of SEQ ID NO: 7 comprising thedeletions T183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 7, or (iv)amino acids 1-485 of SEQ ID NO: 7 comprising the deletions T183*+G184*.

Embodiment 27

The variant of Embodiment 18, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to (i) the mature polypeptide of SEQID NO: 8, (ii) the mature polypeptide of SEQ ID NO: 8 comprising thedeletions D183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 8, or (iv)amino acids 1-485 of SEQ ID NO: 8 comprising the deletions D183*+G184*.

Embodiment 28

The variant of Embodiment 18, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to (i) the mature polypeptide of SEQID NO: 9, (ii) the mature polypeptide of SEQ ID NO: 9 comprising thedeletions D183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 9, or (iv)amino acids 1-485 of SEQ ID NO: 9 comprising the deletions D183*+G184*.

Embodiment 29

The variant of Embodiment 18, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to (i) the mature polypeptide of SEQID NO: 10, (ii) the mature polypeptide of SEQ ID NO: 10 comprising thedeletions D183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 10, or (iv)amino acids 1-485 of SEQ ID NO: 10 comprising the deletions D183*+G184*.

Embodiment 30

The variant of Embodiment 18, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to (i) the mature polypeptide of SEQID NO: 11, (ii) the mature polypeptide of SEQ ID NO: 11 comprising thedeletions H183*+G184*, (iii) amino acids 1-485 of SEQ ID NO: 11, or (iv)amino acids 1-485 of SEQ ID NO: 11 comprising the deletions H183*+G184*.

Embodiment 31

The variant of Embodiment 18, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to (i) the mature polypeptide of SEQID NO: 12, or (ii) amino acids 1-480 of SEQ ID NO: 12.

Embodiment 32

The variant of Embodiment 18, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to (i) the mature polypeptide of SEQID NO: 13, or (ii) amino acids 1-483 of SEQ ID NO: 13.

Embodiment 33

The variant of Embodiment 18, wherein the parent alpha-amylase has atleast 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% sequence identity to (i) the mature polypeptide of SEQID NO: 14, or (ii) amino acids 1-481 of SEQ ID NO: 14.

Embodiment 34

The variant of Embodiment 18, wherein the parent alpha-amylase comprisesor consists of (i) the mature polypeptide of any of SEQ ID NOs: 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, or (ii) amino acids 1 to 483 ofSEQ ID NO: 1, amino acids 1 to 483 of SEQ ID NO: 2, amino acids 1 to 485of SEQ ID NO: 3, amino acids 1 to 482 of SEQ ID NO: 4, amino acids 1 to484 of SEQ ID NO: 5, amino acids 1 to 483 of SEQ ID NO: 6, amino acids 1to 485 of SEQ ID NO: 7, amino acids 1 to 485 of SEQ ID NO: 8, aminoacids 1 to 485 of SEQ ID NO: 9, amino acids 1 to 485 of SEQ ID NO: 10,amino acids 1 to 485 of SEQ ID NO: 11, amino acids 1 to 480 of SEQ IDNO: 12, amino acids 1 to 483 of SEQ ID NO: 13 or amino acids 1 to 481 ofSEQ ID NO: 14.

Embodiment 35

The variant of Embodiment 18, wherein the parent alpha-amylase is afragment of the mature polypeptide of any of SEQ ID NOs: 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13 or 14, wherein the fragment has alpha-amylaseactivity.

Embodiment 36

The variant of any of Embodiments 1-35, wherein the variant consists of300 to 700, e.g., 350 to 650, 400 to 600, 450 to 500 or 470 to 490,amino acids.

Embodiment 37

The variant of any of Embodiments 1-36, which is an isolatedalpha-amylase variant.

Embodiment 38

The variant of any of Embodiments 1-37, which comprises one or moresubstitutions selected from the group consisting of M15T, M15S, M15L,G48A, T49H, T49G, T49L, T49I, S50T, G107A, T116G, H133Y, W138Y, W138F,H156Y, K176L, A181T, A181E, A181D, S187D, N188S, N188T, N190F, M197S,M197T, M197V, M197L, M197I, I201Y, I201F, H205Y, A209V, K213T, S239Q,S239E, S239N, S239D, S239A, S239M, S239C, L241D, E255P, E255G, Q264S,G299R, Q360S, R375V, R375G, D416V, R437W, G474K, G474R, G474E, G474Q,G475K, G475R, G475E and G475Q.

Embodiment 39

The variant of any of Embodiments 1-38, which comprises a substitutionat a position corresponding to position 176.

Embodiment 40

The variant of any of Embodiments 1-39, which comprises the substitutionK176L.

Embodiment 41

The variant of Embodiment 39 or 40, which further comprises asubstitution at one or more positions corresponding to positions 15, 48,49, 50, 107, 116, 133, 138, 156, 181, 187, 188, 190, 197, 201, 205, 209,213, 239, 241, 255, 264, 299, 360, 375, 416, 437, 474 and 475.

Embodiment 42

The variant of Embodiment 40, which further comprises one or moresubstitutions selected from the group consisting of M15T, M15S, M15L,G48A, T49H, T49G, T49L, T49I, S50T, G107A, T116G, H133Y, W138Y, W138F,H156Y, A181T, A181E, A181D, S187D, N188S, N188T, N190F, M197S, M197T,M197V, M197L, M197I, I201Y, I201F, H205Y, A209V, K213T, S239Q, S239E,S239N, S239D, S239A, S239M, S239C, L241D, E255P, E255G, Q264S, G299R,Q360S, R375V, R375G, D416V, R437W, G474K, G474R, G474E, G474Q, G475K,G475R, G475E and G475Q.

Embodiment 43

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 15.

Embodiment 44

The variant of any of Embodiments 1-42, which comprises the substitutionM15T.

Embodiment 45

The variant of any of Embodiments 1-42, which comprises the substitutionM15S.

Embodiment 46

The variant of any of Embodiments 1-42, which comprises the substitutionM15L.

Embodiment 47

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 48.

Embodiment 48

The variant of any of Embodiments 1-42, which comprises the substitutionG48A.

Embodiment 49

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 49.

Embodiment 50

The variant of any of Embodiments 1-42, which comprises the substitutionT49H.

Embodiment 51

The variant of any of Embodiments 1-42, which comprises the substitutionT49G.

Embodiment 52

The variant of any of Embodiments 1-42, which comprises the substitutionT49L.

Embodiment 53

The variant of any of Embodiments 1-42, which comprises the substitutionT49I.

Embodiment 54

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 50.

Embodiment 55

The variant of any of Embodiments 1-42, which comprises the substitutionS50T.

Embodiment 56

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 107.

Embodiment 57

The variant of any of Embodiments 1-42, which comprises the substitutionG107A.

Embodiment 58

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position T116.

Embodiment 59

The variant of any of Embodiments 1-42, which comprises the substitutionT116G.

Embodiment 60

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 133.

Embodiment 61

The variant of any of Embodiments 1-42, which comprises the substitutionH133Y.

Embodiment 62

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 138.

Embodiment 63

The variant of any of Embodiments 1-42, which comprises the substitutionW138Y.

Embodiment 64

The variant of any of Embodiments 1-42, which comprises the substitutionW138F.

Embodiment 65

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 156.

Embodiment 66

The variant of any of Embodiments 1-42, which comprises the substitutionH156Y.

Embodiment 67

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 181.

Embodiment 68

The variant of any of Embodiments 1-42, which comprises the substitutionA181T.

Embodiment 69

The variant of any of Embodiments 1-42, which comprises the substitutionA181E.

Embodiment 70

The variant of any of Embodiments 1-42, which comprises the substitutionA181D.

Embodiment 71

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 187.

Embodiment 72

The variant of any of Embodiments 1-42, which comprises the substitutionS187D.

Embodiment 73

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 188.

Embodiment 74

The variant of any of Embodiments 1-42, which comprises the substitutionN188S.

Embodiment 75

The variant of any of Embodiments 1-42, which comprises the substitutionN188T.

Embodiment 76

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 190.

Embodiment 77

The variant of any of Embodiments 1-42, which comprises the substitutionN190F.

Embodiment 78

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 197.

Embodiment 79

The variant of any of Embodiments 1-42, which comprises the substitutionM197S.

Embodiment 80

The variant of any of Embodiments 1-42, which comprises the substitutionM197T.

Embodiment 81

The variant of any of Embodiments 1-42, which comprises the substitutionM197V.

Embodiment 82

The variant of any of Embodiments 1-42, which comprises the substitutionM197L.

Embodiment 83

The variant of any of Embodiments 1-42, which comprises the substitutionM197I.

Embodiment 84

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 201.

Embodiment 85

The variant of any of Embodiments 1-42, which comprises the substitution1201Y.

Embodiment 86

The variant of any of Embodiments 1-42, which comprises the substitutionI201F.

Embodiment 87

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 205.

Embodiment 88

The variant of any of Embodiments 1-42, which comprises the substitutionH205Y.

Embodiment 89

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 209.

Embodiment 90

The variant of any of Embodiments 1-42, which comprises the substitutionA209V.

Embodiment 91

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 213.

Embodiment 92

The variant of any of Embodiments 1-42, which comprises the substitutionK213T.

Embodiment 93

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 239.

Embodiment 94

The variant of any of Embodiments 1-42, which comprises the substitutionS239Q.

Embodiment 95

The variant of any of Embodiments 1-42, which comprises the substitutionS239E.

Embodiment 96

The variant of any of Embodiments 1-42, which comprises the substitutionS239N.

Embodiment 97

The variant of any of Embodiments 1-42, which comprises the substitutionS239D.

Embodiment 98

The variant of any of Embodiments 1-42, which comprises the substitutionS239A.

Embodiment 99

The variant of any of Embodiments 1-42, which comprises the substitutionS239M.

Embodiment 100

The variant of any Embodiments 1-42, which comprises the substitutionS239C.

Embodiment 101

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 241.

Embodiment 102

The variant of any of Embodiments 1-42, which comprises the substitutionL241D.

Embodiment 103

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 255.

Embodiment 104

The variant of any of Embodiments 1-42, which comprises the substitutionE255P.

Embodiment 105

The variant of any of Embodiments 1-42, which comprises the substitutionE255G.

Embodiment 106

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 264.

Embodiment 107

The variant of any of Embodiments 1-42, which comprises the substitutionQ264S.

Embodiment 108

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 299.

Embodiment 109

The variant of any of Embodiments 1-42, which comprises the substitutionG299R.

Embodiment 110

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 360.

Embodiment 111

The variant of any of Embodiments 1-42, which comprises the substitutionQ360S.

Embodiment 112

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 375.

Embodiment 113

The variant of any of Embodiments 1-42, which comprises the substitutionR375V.

Embodiment 114

The variant of any of Embodiments 1-42, which comprises the substitutionR375G.

Embodiment 115

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 416.

Embodiment 116

The variant of any of Embodiments 1-42, which comprises the substitutionD416V.

Embodiment 117

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 437.

Embodiment 118

The variant of any of Embodiments 1-42, which comprises the substitutionR437W.

Embodiment 119

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 474.

Embodiment 120

The variant of any of Embodiments 1-42, which comprises the substitutionG474K.

Embodiment 121

The variant of any of Embodiments 1-42, which comprises the substitutionG474R.

Embodiment 122

The variant of any of Embodiments 1-42, which comprises the substitutionG474E.

Embodiment 123

The variant of any of Embodiments 1-42, which comprises the substitutionG474Q.

Embodiment 124

The variant of any of Embodiments 1-42, which comprises a substitutionat a position corresponding to position 475.

Embodiment 125

The variant of any of Embodiments 1-42, which comprises the substitutionG475K.

Embodiment 126

The variant of any of Embodiments 1-42, which comprises the substitutionG475R.

Embodiment 127

The variant of any of Embodiments 1-42, which comprises the substitutionG475E.

Embodiment 128

The variant of any of Embodiments 1-42, which comprises the substitutionG475Q.

Embodiment 129

The variant of any of Embodiments 1-42, which comprises a set ofsubstitutions selected from the group consisting of:

T49H+K176L+E185P,

T49G+K176L+E185P,

T49L+S50T+K176L+E185P,

T116G+K176L+E185P,

K176L+E185P,

K176L+E185P+1201Y+H205Y+K213T+Q360S+D416V+R437W,

K176L+E185P+L241D,

K176L+E185P+R375V, and

K176L+E185P+R375G.

Embodiment 130

The variant of any of Embodiments 1-42, which comprises a set ofsubstitutions selected from the group consisting of:

G48A+T49H+G107A+H156Y+K176L+A181T+E185P+N190F+1201F+A209V+Q264S;

G48A+T49G+G107A+H156Y+K176L+A181T+E185P+N190F+1201F+A209V+Q264S;

G48A+T49L+S50T+G107A+H156Y+K176L+A181T+E185P+N190F+1201F+A209V+Q264S;

G48A+T49I+G107A+T116G+H156Y+K176L+A181T+E185P+N190F+1201F+A209V+Q264S;

G48A+T49I+G107A+H156Y+K176L+A181T+E185P+N190F+1201F+A209V+Q264S;

G48A+T49I+G107A+H156Y+K176L+A181T+E185P+N190F+I201Y+H205Y+A209V+K213T+Q264S+Q360S+D416V+R437W;

G48A+T49I+G107A+H156Y+K176L+A181T+E185P+N190F+I201F+L241D+A209V+Q264S;

G48A+T49I+G107A+H156Y+K176L+A181T+E185P+N190F+1201F+A209V+Q264S+R375V;

G48A+T49I+G107A+H156Y+K176L+A181T+E185P+N190F+1201F+A209V+Q264S+R375G;and

G48A+G107A+H156Y+K176L+A181T+E185P+N190F+1201F+A209V+Q264S.

Embodiment 131

The variant of any of Embodiments 1-42, which comprises a substitutionat one or more positions corresponding to positions 49, 50, 116, 176,201, 205, 213, 241, 360, 375, 416, and 437.

Embodiment 132

The variant of Embodiment 131, which comprises one or more substitutionsselected among T49H, T49G, T49L, S50T, T116G, K176L, 1201Y, H205Y,K213T, L241D, Q360S, R375V, R375G, D416V and R437W.

Embodiment 133

The variant of Embodiment 131 or 132, which further comprises asubstitution at one or more positions corresponding to positions 15, 48,107, 133, 138, 156, 181, 187, 188, 190, 197, 209, 239, 255, 264, 299,474 and 475.

Embodiment 134

The variant of Embodiment 133, which comprises one or more substitutionsselected among M15T, M15S, M15L, G48A, G107A, H133Y, W138Y, W138F,H156Y, A181T, A181E, A181D, S187D, N188S, N188T, N190F, M197S, M197T,M197V, M197L, M197I, A209V, S239Q, S239E, S239N, S239D, S239A, S239M,S239C, E255P, E255G, Q264S, G299R, G474K, G474R, G474E, G474Q, G475K,G475R, G475E and G475Q.

Embodiment 135

The variant of any of Embodiments 1-134 which further comprises adeletion at both of the two positions immediately before position 180 ofSEQ ID NO: 1.

Embodiment 136

The variant of any of Embodiments 1-135, wherein the total number ofsubstitutions is 2-20, e.g., 2-10 or 2-5, such as 2, 3, 4, 5, 6, 7, 8, 9or 10 substitutions.

Embodiment 137

The variant of any of Embodiments 1-136, which has increasedthermostability compared to the mature polypeptide of SEQ ID NO: 1 whenincubated at high temperature, low calcium and low pH.

Embodiment 138

The variant of any of Embodiments 1-136, which has increasedthermostability compared to the mature polypeptide of SEQ ID NO: 14 whenincubated at high temperature, low calcium and low pH

Embodiment 139

The variant of any of Embodiments 1-136, which has increasedthermostability compared to its parent when incubated at hightemperature, low calcium and low pH.

Embodiment 140

The variant of any of Embodiments 1-136, which has an increased residualactivity half-life compared to the mature polypeptide of SEQ ID NO: 1when incubated with 0.125 mM CaCl₂ at 90° C. and pH 4.5.

Embodiment 141

The variant of any of Embodiments 1-136, which has an increased residualactivity half-life compared to the mature polypeptide of SEQ ID NO: 14when incubated with 0.125 mM CaCl₂ at 90° C. and pH 4.5.

Embodiment 142

The variant of any of Embodiments 1-136, which has an increased residualactivity half-life compared to its parent when incubated with 0.125 mMCaCl₂ at 90° C. and pH 4.5.

Embodiment 143

A detergent composition comprising the variant of any of Embodiments1-142 and a surfactant.

Embodiment 144

A composition comprising the variant of any of Embodiments 1-142 and oneor more enzymes selected from the group consisting of beta-amylase,cellulase (beta-glucosidase, cellobiohydrolase, and endoglucanase)glucoamylase, hemicellulase (e.g., xylanase), isoamylase, isomerase,lipase, phytase, protease and pullulanase.

Embodiment 145

Use of the variant of any of Embodiments 1-142 for washing and/ordishwashing.

Embodiment 146

Use of the variant of any of Embodiments 1-142 for desizing a textile.

Embodiment 147

Use of the variant of any of Embodiments 1-142 for producing a bakedproduct.

Embodiment 148

Use of the variant of any of Embodiments 1-142 for liquefying astarch-containing material.

Embodiment 149

A method of producing liquefied starch, comprising liquefying astarch-containing material with the variant of any of Embodiments 1-142.

Embodiment 150

A method of producing a fermentation product, comprising

(a) liquefying a starch-containing material with the variant of any ofEmbodiments 1-142 to produce a liquefied mash;

(b) saccharifying the liquefied mash to produce fermentable sugars; and

(c) fermenting the fermentable sugars in the presence of a fermentingorganism.

Embodiment 151

The method of Embodiment 150, wherein step (a) is performed at pH 4-5.

Embodiment 152

A method of producing a fermentation product, comprising contacting astarch substrate with the variant of any of Embodiments 1-142, aglucoamylase, and a fermenting organism.

Embodiment 153

An isolated polynucleotide encoding the variant of any of Embodiments1-142.

Embodiment 154

A nucleic acid construct comprising the polynucleotide of Embodiment153.

Embodiment 155

An expression vector comprising the polynucleotide of Embodiment 153.

Embodiment 156

A host cell comprising the polynucleotide of Embodiment 153.

Embodiment 157

A method of producing an alpha-amylase variant, comprising:

a. cultivating the host cell of Embodiment 156 under conditions suitablefor expression of the variant; and

b. recovering the variant.

Embodiment 158

A method for obtaining an alpha-amylase variant, comprising (a)introducing into a parent alpha-amylase a substitution with proline at aposition corresponding to position 185 of SEQ ID NO: 1 and asubstitution at one or more positions corresponding to positions 15, 48,49, 50, 107, 116, 133, 138, 156, 176, 181, 187, 188, 190, 197, 201, 205,209, 213, 239, 241, 255, 264, 299, 360, 375, 416, 437, 474 and 475 ofSEQ ID NO: 1; and (b) recovering the variant.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

Examples

Assays for Measurement of Amylolytic Activity (Alpha-Amylase Activity)

PNP-G7 Assay:

The alpha-amylase activity is determined by a method employing thePNP-G7 substrate. PNP-G7 is an abbreviation for4,6-ethylidene(G₇)-p-nitrophenyl(G₁)-α,D-maltoheptaoside, a blockedoligosaccharide which can be cleaved by an endo-amylase, such as analpha-amylase. Following the cleavage, the alpha-glucosidase included inthe kit digest the hydrolyzed substrate further to liberate a free PNPmolecule which has a yellow color and thus can be measured by visiblespectophometry at λ=405 nm (400-420 nm). Kits containing PNP-G7substrate and alpha-glucosidase is manufactured by Roche/Hitachi (cat.No. 11876473).

Reagents:

The G7-PNP substrate from this kit contains 22 mM 4,6-ethylidene-G7-PNPand 52.4 mM HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonicacid), pH 7.0.

The alpha-glucosidase reagent contains 52.4 mM HEPES, 87 mM NaCl, 12.6mM MgCl₂, 0.075 mM CaCl₂, >4 kU/L alpha-glucosidase.

The substrate working solution is made by mixing 1 ml of thealpha-glucosidase reagent with 0.2 ml of the G7-PNP substrate. Thissubstrate working solution is made immediately before use.

Dilution buffer: 50 mM EPPS, 0.01% (w/v) Triton X100 (polyethyleneglycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (C₁₄H₂₂O(C₂H₄O),(n=9-10))), 1 mM CaCl₂, pH 7.0.

Procedure:

The amylase sample to be analyzed is diluted in dilution buffer toensure the pH in the diluted sample is 7. The assay is performed bytransferring 20 μl diluted enzyme samples to 96 well microtiter plateand adding 80 μl substrate working solution. The solution is mixed andpre-incubated 1 minute at room temperature and absorption is measuredevery 20 sec. over 5 minutes at OD 405 nm.

The slope (absorbance per minute) of the time dependent absorption-curveis directly proportional to the specific activity (activity per mgenzyme) of the alpha-amylase in question under the given set ofconditions. The amylase sample should be diluted to a level where theslope is below 0.4 absorbance units per minute.

Phadebas Activity Assay:

The alpha-amylase activity can also be determined by a method using thePhadebas substrate (from for example Magle Life Sciences, Lund, Sweden).A Phadebas tablet includes interlinked starch polymers that are in theform of globular microspheres that are insoluble in water. A blue dye iscovantly bound to these microspheres. The interlinked starch polymers inthe microsphere are degraded at a speed that is proportional to thealpha-amylase activity. When the alpha-amylase degrades the starchpolymers, the released blue dye is water soluble and concentration ofdye can be determined by measuring absorbance at 620 nm. Theconcentration of blue is proportional to the alpha-amylase activity inthe sample.

The amylase sample to be analysed is diluted in activity buffer with thedesired pH. One substrate tablet is suspended in 5 mL activity bufferand mixed on magnetic stirrer. During mixing of substrate transfer 150μl to microtiter plate (MTP) or PCR-MTP. Add 30 μl diluted amylasesample to 150 μl substrate and mix. Incubate for 15 minutes at 37° C.The reaction is stopped by adding 30 μl 1 M NaOH and mix. Centrifuge MTPfor 5 minutes at 4000×g. Transfer 100 μl to new MTP and measureabsorbance at 620 nm.

The amylase sample should be diluted so that the absorbance at 620 nm isbetween 0 and 2.2, and is within the linear range of the activity assay.

Reducing Sugar Activity Assay:

The alpha-amylase activity can also be determined by reducing sugarassay with for example corn starch substrate. The number of reducingends formed by the alpha-amylase hydrolyzing the alpha-1,4-glycosidiclinkages in starch is determined by reaction with p-Hydroxybenzoic acidhydrazide (PHBAH). After reaction with PHBAH the number of reducing endscan be measured by absorbance at 405 nm and the concentration ofreducing ends is proportional to the alpha-amylase activity in thesample.

The corns starch substrate (3 mg/ml) is solubilised by cooking for 5minutes in milliQ water and cooled down before assay. For the stopsolution prepare a Ka-Na-tartrate/NaOH solution (K—Na-tartrate (Merck8087) 50 g/l, NaOH 20 g/l) and prepare freshly the stop solution byadding p-Hydroxybenzoic acid hydrazide (PHBAH, Sigma H9882) toKa-Na-tartrate/NaOH solution to 15 mg/ml.

In PCR-MTP 50 μl activity buffer is mixed with 50 μl substrate. Add 50μl diluted enzyme and mix. Incubate at the desired temperature in PCRmachine for 5 minutes. Reaction is stopped by adding 75 μl stop solution(Ka-Na-tartrate/NaOH/PHBAH). Incubate in PCR machine for 10 minutes at95° C. Transfer 150 μl to new MTP and measure absorbance at 405 nm.

The amylase sample should be diluted so that the absorbance at 405 nm isbetween 0 and 2.2, and is within the linear range of the activity assay.

Enzchek® Assay:

For the determination of residual amylase activity an EnzChek® UltraAmylase Assay Kit (E33651, Invitrogen, La Jolla, Calif., USA) was used.

The substrate is a corn starch derivative, DQ™ starch, which is cornstarch labeled with BODIPY® FL dye to such a degree that fluorescence isquenched. One vial containing approx. 1 mg lyophilized substrate isdissolved in 100 microliters of 50 mM sodium acetate (pH 4.0). The vialis vortexed for 20 seconds and left at room temperature, in the dark,with occasional mixing until dissolved. Then 900 microliters of 100 mMacetate, 0.01% (w/v) TRITON® X100, 0.125 mM CaCl₂, pH 5.5 is added,vortexed thoroughly and stored at room temperature, in the dark untilready to use. The stock substrate working solution is prepared bydiluting 10-fold in residual activity buffer (100 mM acetate, 0.01%(w/v) TRITON® X100, 0.125 mM CaCl₂, pH 5.5). Immediately afterincubation the enzyme is diluted to a concentration of 10-20 ng enzymeprotein/ml in 100 mM acetate, 0.01% (W/v) TRITON® X100, 0.125 mM CaCl₂,pH 5.5.

For the assay, 25 microliters of the substrate working solution is mixedfor 10 second with 25 microliters of the diluted enzyme in a black 384well microtiter plate. The fluorescence intensity is measured(excitation: 485 nm, emission: 555 nm) once every minute for 15 minutesin each well at 25° C. and the V_(max) is calculated as the slope of theplot of fluorescence intensity against time. The plot should be linearand the residual activity assay has been adjusted so that the dilutedreference enzyme solution is within the linear range of the activityassay.

Reference Alpha-Amylase and Variants Thereof

The reference alpha-amylase is LE399 (previously disclosed in, e.g., WO02/10355). LE399 comprises amino acids 1-37 of the alpha-amylase fromBacillus amyloliquefaciens (SEQ ID NO: 6) and amino acids 40-483 of thealpha-amylase from Bacillus licheniformis (SEQ ID NO: 1) with thefollowing substitutions G48A T49I G107A H156Y A181T N190F1201F A209VQ264S.

The variants tested are variants of LE399, so the substitutions in eachvariant as listed in the tables below are substitutions as compared toLE399. The position numbering is according to SEQ ID NO: 1.

Example 1

Thermostability of Alpha-Amylase Variants at pH 4.5

The thermostability of a reference alpha-amylase and alpha-amylasevariants thereof was determined by incubating the referencealpha-amylase and variants at pH 4.5 and temperatures of 90° C. and 95°C. with 0.125 mM CaCl₂ followed by residual activity determination usingthe EnzChek® substrate (EnzChek® Ultra Amylase assay kit, E33651,Molecular Probes).

Purified enzyme samples were diluted to working concentrations of 5 and10 ppm (micrograms/ml) in enzyme dilution buffer (10 mM acetate, 0.01%Triton X100, 0.125 mM CaCl₂, pH 5.0). Twenty microliters enzyme samplewas transferred to 96-well PCR MTP and 180 microliters stability buffer(150 mM acetate, 0.01% Triton X100, 0.125 mM CaCl₂, pH 4.5 or 4.8) wasadded to each well and mixed. The assay was performed using twoconcentrations of enzyme in duplicates. Before incubation at 90° C. or95° C., 20 microliters was withdrawn and stored on ice as unstressedcontrol samples. Incubation was performed in a PCR machine for 2 or 8minutes (pH 4.5 and 90° C.) and 30 minutes (pH 4.5 and 95° C.).Incubation time (minutes) is selected so that residual activity isbetween 10-90%.

After incubation samples were diluted to 15 ng/ml in residual activitybuffer (100 mM Acetate, 0.01% Triton X100, 0.125 mM CaCl₂, pH 5.5) and25 microliters diluted enzyme was transferred to black 384-well MTP.Residual activity was determined using the EnzChek® substrate by adding25 microliters substrate solution (100 micrograms/ml) to each well.Fluorescence was determined at 25° C. every minute for 15 minutes usingexcitation filter at 485-P nm and emission filter at 555 nm(fluorescence reader is Polarstar, BMG). The residual activity wasnormalized to unstressed control samples for each setup.

Assuming exponential decay half life time (T½ (min)) was calculatedusing the equation: T½ (min)=T(min)*LN(0.5)/LN(% RA/100), where T isassay incubation time in minutes, and % RA is % residual activitydetermined in assay.

Using this assay setup the half life time was determined as a measure ofthermostability for the reference alpha-amylase and variants thereof asshown in Tables 1 and 2.

The reference amylase was not tested at pH 4.5, 0.125 mM CaCl₂) at 95°C. for 30 minutes since there is no residual activity (half life time is1.2 minutes at 90° C.).

TABLE 1 Conditions: pH 4.5, 0.125 mM CaCl₂, 90° C. for 2 or 8 minutesT½(min) (pH 4.5, 0.125 Mutations mM CaCl₂, 90° C.) Reference amylase 1.2K176L F201Y H205Y K213T 8 Q360S D416V R437W K176L E185P F201Y H205Y 54K213T Q360S D416V R437W K176L 6 E185P 26 K176L E185P 100% residualactivity

TABLE 2 Conditions: pH 4.5, 0.125 mM CaCl₂, 95° C. for 30 minutesT½(min) (pH 4.5, 0.125 Mutations mM CaCl₂, 95° C.) K176L E185P 20 T116GK176L E185P 23

Example 2

Thermostability of Alpha-Amylase Variants at pH 4.8

The thermostability of a reference alpha-amylase and alpha-amylasevariants thereof was determined by incubating the referencealpha-amylase and variants at pH 4.8 and temperatures of 90° C. and 95°C. with 0.125 mM CaCl₂) followed by residual activity determinationusing the EnzChek® substrate (EnzChek® Ultra Amylase assay kit, E33651,Molecular Probes).

Purified enzyme samples were diluted to working concentrations of 5 and10 ppm (micrograms/ml) in enzyme dilution buffer (10 mM acetate, 0.01%Triton X100, 0.125 mM CaCl₂, pH 5.0). Twenty microliters enzyme samplewas transferred to 96-well PCR MTP and 180 microliters stability buffer(150 mM acetate, 0.01% Triton X100, 0.125 mM CaCl₂, pH 4.5 or 4.8) wasadded to each well and mixed. The assay was performed using twoconcentrations of enzyme in duplicates. Before incubation at 90° C. or95° C., 20 microliters was withdrawn and stored on ice as unstressedcontrol samples. Incubation was performed in a PCR machine for 12, 20,or 25 minutes (pH 4.8 and 90° C.) or 50 minutes (pH 4.8 and 95° C.).Incubation time (minutes) is selected so that residual activity isbetween 10-90%.

After incubation samples were diluted to 10-20 ng/ml in residualactivity buffer (100 mM Acetate, 0.01% Triton X100, 0.125 mM CaCl₂, pH5.5) and 25 microliters diluted enzyme was transferred to black 384-wellMTP. Residual activity was determined using the EnzChek® substrate byadding 25 microliters substrate solution (100 micrograms/ml) to eachwell. Fluorescence was determined at 25° C. every minute for 15 minutesusing excitation filter at 485-P nm and emission filter at 555 nm(fluorescence reader is Polarstar, BMG). The residual activity wasnormalized to control samples for each setup.

Assuming exponential decay half life time (T½ (min)) was calculatedusing the equation: T½ (min)=T(min)*LN(0.5)/LN(% RA/100), where T isassay incubation time in minutes, and % RA is % residual activitydetermined in assay.

Using this assay setup the half life time was determined as a measure ofthermostability for the reference alpha-amylase and variants thereof asshown in Tables 3 and 4.

The reference amylase was not tested at pH 4.8, 0.125 mM CaCl₂) at 95°C. for 50 minutes since there is no residual activity (half life time is4.6 minutes at 90° C.).

TABLE 3 Conditions: pH 4.8, 0.125 mM CaCl₂, 90° C. for 12, 20 or 25minutes T½(min) (pH 4.8, 0.125 Mutations mM CaCl₂, 90° C.) Referenceamylase 4.6 K176L F201Y H205Y K213T 25 Q360S D416V R437W K176L E185PF201Y H205Y 53 K213T Q360S D416V R437W K176L 21 E185P 75 K176L E185P 127

TABLE 4 Conditions: pH 4.8, 0.125 mM CaCl₂, 95° C. for 50 minutesT½(min) (pH 4.8, 0.125 Mutations mM CaCl₂, 95° C.) K176L E185P 42 T116GK176L E185P 55 K176L E185P L241D 49 K176L E185P R375V 52 K176L E185PR375G 51 I49T K176L E185P 47 I49H K176L E185P 48 I49G K176L E185P 43I49L S50T K176L E185P 48

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

The invention claimed is:
 1. An alpha-amylase variant comprising asubstitution at position 185 with proline and a substitution at one ormore positions 15, 48, 49, 50, 107, 116, 133, 138, 156, 181, 187, 188,190, 197, 205, 209, 213, 241, 255, 264, 299, 360, 375, 416, 437, 474 and475, wherein (a) the variant has at least 80% and less than 100%sequence identity to: (i) amino acids 1 to 483 of SEQ ID NO: 1, (ii)amino acids 1 to 483 of SEQ ID NO: 2, (iii) amino acids 1 to 485 of SEQID NO: 3, (iv) amino acids 1 to 482 of SEQ ID NO: 4, (v) amino acids 1to 484 of SEQ ID NO: 5, (vi) amino acids 1 to 483 of SEQ ID NO: 6, (vii)amino acids 1 to 485 of SEQ ID NO: 7, (viii) amino acids 1 to 485 of SEQID NO: 8, (ix) amino acids 1 to 485 of SEQ ID NO: 9, (x) amino acids 1to 485 of SEQ ID NO: 10, (xi) amino acids 1 to 485 of SEQ ID NO: 11,(xii) amino acids 1 to 480 of SEQ ID NO: 12, (xiii) amino acids 1 to 483of SEQ ID NO: 13, or (xiv) amino acids 1 to 481 of SEQ ID NO: 14, (b)each position corresponds to the position of the alpha-amylase of SEQ IDNO: 1, and (c) the variant has alpha-amylase activity.
 2. The variant ofclaim 1, which comprises: a substitution at position 15 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr,Trp, Tyr or Val; a substitution at position 48 with Ala, Arg, Asn, Asp,Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr orVal; a substitution at position 49 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val; asubstitution at position 50 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr or Val; a substitutionat position 107 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; a substitution atposition 116 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val; a substitution at position 133with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr or Val; a substitution at position 138 with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Tyr or Val; a substitution at position 156 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr or Val; a substitution at position 181 with Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val;a substitution at position 187 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr or Val; asubstitution at position 188 with Ala, Arg, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; asubstitution at position 190 with Ala, Arg, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; asubstitution at position 197 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; asubstitution at position 205 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; asubstitution at position 209 with Arg, Asn, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; asubstitution at position 213 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; asubstitution at position 241 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; asubstitution at position 255 with Ala, Arg, Asn, Asp, Cys, Gln, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; asubstitution at position 264 with Ala, Arg, Asn, Asp, Cys, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; asubstitution at position 299 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; asubstitution at position 360 with Ala, Arg, Asn, Asp, Cys, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; asubstitution at position 375 with Ala, Asn, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; asubstitution at position 416 with Ala, Arg, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; asubstitution at position 437 with Ala, Asn, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; asubstitution at position 474 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; or asubstitution at position 475 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val.
 3. Thevariant of claim 1, which comprises: a substitution at position 15 withLeu, Ser or Thr; a substitution at position 48 with Ala; a substitutionat position 49 with Gly, His, Ile or Leu; a substitution at position 50with Thr; a substitution at position 107 with Ala; a substitution atposition 116 with Gly; a substitution at position 133 with Tyr; asubstitution at position 138 with Phe or Tyr; a substitution at position156 with Tyr; a substitution at position 181 with Asp, Glu or Thr; asubstitution at position 187 with Asp; a substitution at position 188with Ser or Thr; a substitution at position 190 with Phe; a substitutionat position 197 with Ile, Leu, Ser, Thr or Val; a substitution atposition 205 with Tyr; a substitution at position 209 with Val; asubstitution at position 213 with Thr; a substitution at position 241with Asp; a substitution at position 255 with Gly or Pro; a substitutionat position 264 with Ser; a substitution at position 299 with Arg; asubstitution at position 360 with Ser; a substitution at position 375with Gly or Val; a substitution at position 416 with Val; a substitutionat position 437 with Trp; a substitution at position 474 with Arg, Gin,Glu or Lys; or a substitution at position 475 with Arg, Gin, Glu or Lys.4. The variant of claim 1, which comprises one or more substitutionsselected from the group consisting of M15T, M15S, M15L, G48A, T49H,T49G, T49L, T49I, S50T, G107A, T116G, H133Y, W138Y, W138F, H156Y, A181T,A181E, A181D, S187D, N188S, N188T, N190F, M197S, M197T, M197V, M197L,M197I, H205Y, A209V, K213T, L241D, E255P, E255G, Q264S, G299R, Q360S,R375V, R375G, D416V, R437W, G474K, G474R, G474E, G474Q, G475K, G475R,G475E and G475Q.
 5. The variant of claim 1, which comprises T116N,T116D, T116E, T116Q, or T116Y.
 6. The variant of claim 1, which furthercomprises one or more of: a substitution at position 176 with Ala, Arg,Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr,Trp, Tyr or Val; a substitution at position 201 with Ala, Arg, Asn, Asp,Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr orVal; and a substitution at position 239 with Ala, Arg, Asn, Asp, Cys,Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr or Val.7. The variant of claim 1, which further comprises one or more of: asubstitution at position 176 with Leu; a substitution at position 201with Phe or Tyr; and a substitution at position 239 with Ala, Asn, Asp,Cys, Gln, Glu or Met.
 8. The variant of claim 2, which has at least 85%sequence identity to amino acids 1 to 483 of SEQ ID NO: 1, amino acids 1to 483 of SEQ ID NO: 2, amino acids 1 to 485 of SEQ ID NO: 3, aminoacids 1 to 482 of SEQ ID NO: 4, amino acids 1 to 484 of SEQ ID NO: 5,amino acids 1 to 483 of SEQ ID NO: 6, amino acids 1 to 485 of SEQ ID NO:7, amino acids 1 to 485 of SEQ ID NO: 8, amino acids 1 to 485 of SEQ IDNO: 9, amino acids 1 to 485 of SEQ ID NO: 10, amino acids 1 to 485 ofSEQ ID NO: 11, amino acids 1 to 480 of SEQ ID NO: 12, amino acids 1 to483 of SEQ ID NO: 13, or amino acids 1 to 481 of SEQ ID NO:
 14. 9. Thevariant of claim 2, which has at least 90% sequence identity to aminoacids 1 to 483 of SEQ ID NO: 1, amino acids 1 to 483 of SEQ ID NO: 2,amino acids 1 to 485 of SEQ ID NO: 3, amino acids 1 to 482 of SEQ ID NO:4, amino acids 1 to 484 of SEQ ID NO: 5, amino acids 1 to 483 of SEQ IDNO: 6, amino acids 1 to 485 of SEQ ID NO: 7, amino acids 1 to 485 of SEQID NO: 8, amino acids 1 to 485 of SEQ ID NO: 9, amino acids 1 to 485 ofSEQ ID NO: 10, amino acids 1 to 485 of SEQ ID NO: 11, amino acids 1 to480 of SEQ ID NO: 12, amino acids 1 to 483 of SEQ ID NO: 13, or aminoacids 1 to 481 of SEQ ID NO:
 14. 10. The variant of claim 2, which hasat least 95% sequence identity to amino acids 1 to 483 of SEQ ID NO: 1,amino acids 1 to 483 of SEQ ID NO: 2, amino acids 1 to 485 of SEQ ID NO:3, amino acids 1 to 482 of SEQ ID NO: 4, amino acids 1 to 484 of SEQ IDNO: 5, amino acids 1 to 483 of SEQ ID NO: 6, amino acids 1 to 485 of SEQID NO: 7, amino acids 1 to 485 of SEQ ID NO: 8, amino acids 1 to 485 ofSEQ ID NO: 9, amino acids 1 to 485 of SEQ ID NO: 10, amino acids 1 to485 of SEQ ID NO: 11, amino acids 1 to 480 of SEQ ID NO: 12, amino acids1 to 483 of SEQ ID NO: 13, or amino acids 1 to 481 of SEQ ID NO:
 14. 11.The variant of claim 1, which has an increased residual activityhalf-life compared to the mature polypeptide of SEQ ID NO: 1 whenincubated with 0.125 mM CaCl₂) at 90° C. and pH 4.5.
 12. Apolynucleotide encoding the variant of claim
 1. 13. A nucleic acidconstruct or expression vector comprising the polynucleotide of claim12.
 14. A host cell comprising the nucleic acid construct or expressionvector of claim
 13. 15. A method of producing an alpha-amylase variant,comprising: a. cultivating the host cell of claim 14 under conditionssuitable for expression of the variant; and b. recovering the variant.16. A method of producing liquefied starch, comprising liquefying astarch-containing material with the variant of claim
 1. 17. A method ofproducing a fermentation product, comprising (a) liquefying astarch-containing material with the variant of claim 1 to produce aliquefied mash; (b) saccharifying the liquefied mash to producefermentable sugars; and (c) fermenting the fermentable sugars in thepresence of a fermenting organism.
 18. The method of claim 17, whereinstep (a) is performed at pH 4-5.
 19. A method of producing afermentation product, comprising contacting a starch substrate with thevariant of claim 1, a glucoamylase, and a fermenting organism.